Compounds

ABSTRACT

p101 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing p101 polypeptides and polynucleotides in therapy, and diagnostic assays for such.

FIELD OF THE INVENTION

This invention relates to newly identified polypeptides andpolynucleotides encoding such polypeptides, to their use in therapy andin identifying compounds which may be agonists, antagonists and/orinhibitors which are potentially useful in therapy, and to production ofsuch polypeptides and polynucleotides.

BACKGROUND OF THE INVENTION

The drug discovery process is currently undergoing a fundamentalrevolution as it embraces ‘functional genomics’, that is, highthroughput genome- or gene-based biology. This approach as a means toidentify genes and gene products as therapeutic targets is rapidlysuperceding earlier approaches based on ‘positional cloning’. Aphenotype, that is a biological function or genetic disease, would beidentified and this would then be tracked back to the responsible gene,based on its genetic map position.

Functional genomics relies heavily on high-throughput DNA sequencingtechnologies and the various tools of bioinformatics to identify genesequences of potential interest from the many molecular biologydatabases now available. There is a continuing need to identify andcharacterise further genes and their related polypeptides/proteins, astargets for drug discovery.

SUMMARY OF THE INVENTION

The present invention relates to p101 polypeptides and polynucleotides,in particular p101 splice variant polypeptides and polynucleotides,recombinant materials and methods for their production. In anotheraspect, the invention relates to methods for using such polypeptides andpolynucleotides, including the treatment of diseases that involveleucocyte activation and infiltration including inflammatory diseasessuch as COPD, ARDS, arthritis, psoriasis and so on, hereinafter referredto as “the Diseases”, amongst others. In a further aspect, the inventionrelates to methods for identifying agonists and antagonists/inhibitorsusing the materials provided by the invention, and treating conditionsassociated with p101 imbalance with the identified compounds. In a stillfurther aspect, the invention relates to diagnostic assays for detectingdiseases associated with inappropriate p101 activity or levels.

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to p101 splice variantpolypeptides. Such peptides include isolated polypeptides comprising anamino acid sequence which has at least 95% identity, preferably at least97-99% identity, to that of SEQ ID NO:2 or SEQ ID NO:4 over the entirelength of SEQ ID NO:2 or SEQ ID NO:4 respectively. Such polypeptidesinclude those comprising the amino acid sequence of SEQ ID NO:2 or SEQID NO:4 respectively.

Further peptides of the present invention include isolated polypeptidesin which the amino acid sequence has at least 95% identity preferably atleast 97-99% identity, to the amino acid sequence of SEQ ID NO:2 or SEQID NO:4 over the entire length of SEQ ID NO:2 or SEQ ID NO:4. Suchpolypeptides include the polypeptides of SEQ ID NO:2 or SEQ ID NO:4respectively.

Further peptides of the present invention include isolated polypeptidesencoded by a polynucleotide comprising the sequence contained in SEQ IDNO: 1 or SEQ ID NO: 3.

Polypeptides of the present invention are believed to be members of theadaptor protein family of polypeptides. They are therefore of interestbecause they are involved in the generation of the important secondmessenger, phosphatidylinositol 3,4,5-triphosphate (PIP3); PIP3 isgenerated following the stimulation of various receptors and isinvolved, for example in leucocytes, in regulating chemotaxis, adherenceand degranulation. PIP3 is primarily generated via the action ofphosphatidylinositol 3-kinase, several of which are thought to exist.however, one that appears to be particularly relevant in leucocytes isdirectly regulated, i.e activated by G protein βγ subunits. Importantly,this regulation is dependent upon an adaptor protein, p101. Inhibitionof this activation process by, for example, preventing Gβγ binding top101 should prevent PIP3 accumulation. Such an action would be ofbenefit in various disease states that involve leucocyte activation andinfiltration. These properties are hereinafter referred to as “p101activity” or “p101 polypeptide activity” or “biological activity ofp101”. Also included amongst these activities are antigenic andimmunogenic activities of said p101 polypeptides, in particular theantigenic and immunogenic activities of the polypeptide of SEQ ID NO:2or SEQ ID NO:4. Preferably, a polypeptide of the present inventionexhibits at least one biological activity of p101.

The polypeptides of the present invention may be in the form of the“mature” protein or may be a part of a larger protein such as aprecursor or a fusion protein. It is often advantageous to include anadditional amino acid sequence which contains secretory or leadersequences, pro-sequences, sequences which aid in purification such asmultiple histidine residues, or an additional sequence for stabilityduring recombinant production.

The present invention also includes variants of the aforementionedpolypeptides, that is polypeptides that vary from the referents byconservative amino acid substitutions, whereby a residue is substitutedby another with like characteristics. Typical such substitutions areamong Ala, Val, Leu and Ile; among Ser and Thr; among the acidicresidues Asp and Glu; among Asn and Gln; and among the basic residuesLys and Arg; or aromatic residues Phe and Tyr. Particularly preferredare variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids aresubstituted, deleted, or added in any combination.

Polypeptides of the present invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

In a further aspect, the present invention relates to p101 splicevariant polynucleotides. Such polynucleotides include isolatedpolynucleotides comprising a nucleotide sequence encoding a polypeptidewhich has at least 95% identity, to the amino acid sequence of SEQ IDNO:2 or SEQ ID NO:4, over the entire length of SEQ ID NO:2 or SEQ IDNO:4 respectively. In this regard, polypeptides which have at least 97%identity are highly preferred, whilst those with at least 98-99%identity are more highly preferred, and those with at least 99% identityare most highly preferred. Such polynucleotides include a polynucleotidecomprising the nucleotide sequence contained in SEQ ID NO: 1 or SEQ IDNO:3 encoding the polypeptide of SEQ ID NO:2 or SEQ ID NO:4respectively.

Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence that has at least 95%identity to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2or SEQ ID NO:4 respectively, over the entire coding region. In thisregard, polynucleotides which have at least 97% identity are highlypreferred, whilst those with at least 98-99% identity are more highlypreferred, and those with at least 99% identity are most highlypreferred.

Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence which has at least 95%identity to SEQ ID NO: 1 or SEQ ID NO:3 over the entire length of SEQ IDNO: 1 or SEQ ID NO:3 respectively. In this regard, polynucleotides whichhave at least 97% identity are highly preferred, whilst those with atleast 98-99% identiy are more highly preferred, and those with at least99% identity are most highly preferred. Such polynucleotides include apolynucleotide comprising the polynucleotide of SEQ ID NO: 1 or SEQ IDNO:3 as well as the polynucleotide of SEQ ID NO: 1 or SEQ ID NO:3respectively. The invention also provides polynucleotides which arecomplementary to all the above described polynucleotides.

The nucleotide sequence of SEQ ID NO:5, the full-length human p101 cDNAsequence (European Patent Application No: EP98306696.0; SmithKlineBeecham), shows homology with pig p101 (L. R. Stephens et al, Cell 89 pp105-114, 1997). The nucleotide sequence of SEQ ID NO:5 is a cDNAsequence and comprises a polypeptide encoding sequence (nucleotide 1 to3630, Exon 1(1-106), Exon 2 (107-205), Exon 3 (206-265), Exon4(266-414), Exon 5 (415-479), Exon 6 (480-648), Exon 7 (649-810), Exon 8(811-894), Exon 9 (895-1616), Exon 10 (1617-1778), Exon 11 (1779-1907),Exon 12 (1908-2037), Exon 13 (2038-2129), Exon 14 (2130-2200), Exon 15(2201-2298), Exon 16 (2299-2380), Exon 17 (2381-2488), Exon 18(2489-2642)) encoding a polypeptide of 880 amino acids, the polypeptideof SEQ ID NO:6.

SEQ ID NO: 1 is a CDNA which encodes a splice variant of p101, SVP-2,which lacks exons 6, 7, 8, 9 and 10. The polypeptide encoded by thepolynucleotide shown in SEQ ID NO: 1 is given in SEQ ID NO:2. SEQ IDNO:3 is a CDNA which encodes a further splice variant of p101, SVP-4,which lacks exons 9 and 10. The polypeptide encoded by thepolynucleotide shown in SEQ ID NO:3 is given in SEQ ID NO:4.

The nucleotide sequence encoding the polypeptide of SEQ ID NO:2 or SEQID NO:4 may be identical to the polypeptide encoding sequence containedin SEQ ID NO: 1 or SEQ ID NO:3 or it may be a sequence other than theone contained in SEQ ID NO: 1 or SEQ ID NO:3 which, as a result of theredundancy (degeneracy) of the genetic code, also encodes thepolypeptide of SEQ ID NO:2 or SEQ ID NO:4. The polypeptide of the SEQ IDNO:2 or SEQ ID NO:4 is structurally related to other proteins of theadaptor protein family, having homology and/or structural similaritywith pig p101 (L. R. Stephens et al, Cell 89 pp105-114, 1997).

Preferred polypeptides and polynucleotides of the present invention areexpected to have, inter alia, similar biological functions/properties totheir homologous polypeptides and polynucleotides. Furthermore,preferred polypeptides and polynucleotides of the present invention haveat least one p101 activity.

The present invention also relates to partial or other polynucleotideand polypeptide sequences which were first identified prior to thedetermination of the corresponding full length sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

Accordingly, in a further aspect, the present invention provides for anisolated polynucleotide which:

(a) comprises a nucleotide sequence which has at least 95% identity,preferably at least 97-99% identity to SEQ ID NO:7 or SEQ ID NO:9 overthe entire length of SEQ ID NO:7 or SEQ ID NO:9;

(b) has a nucleotide sequence which has at least 95% identity,preferably at least 97-99% identity, to SEQ ID NO:7 or SEQ ID NO:9 overthe entire length of SEQ ID NO:7 or SEQ ID NO:9;

(c) the polynucleotide of SEQ ID NO:7 or SEQ ID NO:9; or

(d) a nucleotide sequence encoding a polypeptide which has at least 95%identity, preferably at least 97-99% identity, to the amino acidsequence of SEQ ID NO:8 or SEQ ID NO: 10 over the entire length of SEQID NO:8 or SEQ ID NO:10;

as well as the polynucleotide of SEQ ID NO:7 or SEQ ID NO:9.

The present invention further provides for a polypeptide which:

(a) comprises an amino acid sequence which has at least 95% identity,preferably at least 97-99% identity, to that of SEQ ID NO:8 or SEQ IDNO: 10 over the entire length of SEQ ID NO:8 or SEQ ID NO: 10;

(b) has an amino acid sequence which is at least 95% identity,preferably at least 97-99% identity, to the amino acid sequence of SEQID NO: 8 or SEQ ID NO: 10 over the entire length of SEQ ID NO:8 or SEQID NO:10;

(c) comprises the amino acid sequence of SEQ ID NO:8 or SEQ ID NO: 10;and

(d) is the polypeptide of SEQ ID NO:8 or SEQ ID NO: 10;

as well as polypeptides encoded by a polynucleotide comprising thesequence contained in SEQ ID NO:7 or SEQ ID NO:9.

The nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:9 and the peptidesequences encoded thereby are derived from EST (Expressed Sequence Tag)sequences. It is recognised by those skilled in the art that there willinevitably be some nucleotide sequence reading errors in EST sequences(see Adams, M. D. et al, Nature 377 (supp) 3, 1995). Accordingly, thenucleotide sequence of SEQ ID NO:7 or SEQ ID NO:9 and the peptidesequence encoded therefrom are therefore subject to the same inherentlimitations in sequence accuracy. Furthermore, the peptide sequenceencoded by SEQ ID NO:7 or SEQ ID NO:9 comprises a region of identity orclose homology and/or close structural similarity (for example aconservative amino acid difference) with the closest homologous orstructurally similar protein.

Polynucleotides of the present invention may be obtained, using standardcloning and screening techniques, from a cDNA library derived from mRNAin cells of human primary monocytes (Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989). Polynucleotides of the invention can also beobtained from natural sources such as genomic DNA libraries or can besynthesized using well known and commercially available techniques.

When polynucleotides of the present invention are used for therecombinant production of polypeptides of the present invention, thepolynucleotide may include the coding sequence for the maturepolypeptide, by itself; or the coding sequence for the maturepolypeptide in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence, or other fusion peptide portions. For example, amarker sequence which facilitates purification of the fused polypeptidecan be encoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., ProcNatl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotidemay also contain non-coding 5′ and 3′ sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

Further embodiments of the present invention include polynucleotidesencoding polypeptide variants which comprise the amino acid sequence ofSEQ ID NO:2, 4, 6 and 8 respectively and in which several, for instancefrom 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid residues aresubstituted, deleted or added, in any combination.

Polynucleotides which are identical or sufficiently identical to anucleotide sequence contained in SEQ ID NO: 1, 3, 7 and 9, may be usedas hybridization probes for cDNA and genomic DNA or as primers for anucleic acid amplification (PCR) reaction, to isolate full-length cDNAsand genomic clones encoding polypeptides of the present invention and toisolate cDNA and genomic clones of other genes (including genes encodingparalogs from human sources and orthologs and paralogs from speciesother than human) that have a high sequence similarity to SEQ ID NO: 1,3, 7 and 9. Typically these nucleotide sequences are 70% identical,preferably 80% identical, more preferably 90% identical, most preferably95% identical to that of the referent. The probes or primers willgenerally comprise at least 15 nucleotides, preferably, at least 30nucleotides and may have at least 50 nucleotides. Particularly preferredprobes will have between 30 and 50 nucleotides. Particularly preferredprimers will have between 20 and 25 nucleotides.

A polynucleotide encoding a polypeptide of the present invention,including homologs from species other than human, may be obtained by aprocess which comprises the steps of screening an appropriate libraryunder stringent hybridization conditions with a labeled probe having thesequence of SEQ ID NO: 1, 3, 7 and 9 respectively or a fragment thereof;and isolating full-length cDNA and genomic clones containing saidpolynucleotide sequence. Such hybridization techniques are well known tothe skilled artisan. Preferred stringent hybridization conditionsinclude overnight incubation at 42° C. in a solution comprising: 50%formamide, 5× SSC (150 nmM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20microgram/ml denatured, sheared salmon sperm DNA; followed by washingthe filters in 0.1× SSC at about 65° C. Thus the present invention alsoincludes polynucleotides obtainable by screening an appropriate libraryunder stingent hybridization conditions with a labeled probe having thesequence of SEQ ID NO:1, 3, 7 and 9 or a fragment thereof.

The skilled artisan will appreciate that, in many cases, an isolatedcDNA sequence will be incomplete, in that the region coding for thepolypeptide is short at the 5′ end of the cDNA. This is a consequence ofreverse transcriptase, an enzyme with inherently low ‘processivity’ (ameasure of the ability of the enzyme to remain attached to the templateduring the polymerisation reaction), failing to complete a DNA copy ofthe mRNA template during 1st strand cDNA synthesis.

There are several methods available and well known to those skilled inthe art to obtain full-length cDNAs, or extend short cDNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman et al., PNAS USA 85, 8998-9002, 1988). Recentmodifications of the technique, exemplified by the Marathon™ technology(Clontech Laboratories Inc.) for example, have significantly simplifiedthe search for longer cDNAs. In the Marathon™ technology, cDNAs havebeen prepared from mRNA extracted from a chosen tissue and an ‘adaptor’sequence ligated onto each end. Nucleic acid amplification (PCR) is thencarried out to amplify the ‘missing’ 5′ end of the cDNA using acombination of gene specific and adaptor specific oligonucleotideprimers. The PCR reaction is then repeated using ‘nested’ primers, thatis, primers designed to anneal within the amplified product (typicallyan adaptor specific primer that anneals further 3′ in the adaptorsequence and a gene specific primer that anneals further 5′ in the knowngene sequence). The products of this reaction can then be analysed byDNA sequencing and a full-length cDNA constructed either by joining theproduct directly to the existing cDNA to give a complete sequence, orcarrying out a separate full-length PCR using the new sequenceinformation for the design of the 5′ primer.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in the art from genetically engineered host cellscomprising expression systems. Accordingly, in a further aspect, thepresent invention relates to expression systems which comprise apolynucleotide or polynucleotides of the present invention, to hostcells which are genetically engineered with such expression sytems andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Introduction of polynucleotides into hostcells can be effected by methods described in many standard laboratorymanuals, such as Davis et al., Basic Methods in Molecular Biology (1986)and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).Preferred such methods include, for instance, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used, for instance,chromosomal, episomal and virus-derived systems, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression systems may containcontrol regions that regulate as well as engender expression. Generally,any system or vector which is able to maintain, propagate or express apolynucleotide to produce a polypeptide in a host may be used. Theappropriate nucleotide sequence may be inserted into an expressionsystem by any of a variety of well-known and routine techniques, suchas, for example, those set forth in Sambrook et al., Molecular Cloning,A Laboratory Manual (supra). Appropriate secretion signals may beincorporated into the desired polypeptide to allow secretion of thetranslated protein into the lumen of the endoplasmic reticulum, theperiplasmic space or the extracellular environment. These signals may beendogenous to the polypeptide or they may be heterologous signals.

If a polypeptide of the present invention is to be expressed for use inscreening assays, it is generally preferred that the polypeptide beproduced at the surface of the cell. In this event, the cells may beharvested prior to use in the screening assay. If the polypeptide issecreted into the medium, the medium can be recovered in order torecover and purify the polypeptide. If produced intracellularly, thecells must first be lysed before the polypeptide is recovered.

Polypeptides of the present invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding proteins may be employed to regenerateactive conformation when the polypeptide is denatured duringintracellular synthesis, isolation and or purification.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of thegene characterised by the polynucleotide of SEQ ID NO: 1, 3, 5 and 7respectively which is associated with a dysfunction will provide adiagnostic tool that can add to, or define, a diagnosis of a disease, orsusceptibility to a disease, which results from under-expression,over-expression or altered spatial or temporal expression of the gene.Individuals carrying mutations in the gene may be detected at the DNAlevel by a variety of techniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR or other amplification techniques prior toanalysis. RNA or cDNA may also be used in similar fashion. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to labeled p101 nucleotide sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase digestion or by differences in melting temperatures.DNA sequence differences may also be detected by alterations inelectrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing (ee, e.g., Myers et al.,Science (1985) 230:1242). Sequence changes at specific locations mayalso be revealed by nuclease protection assays, such as RNase and S1protection or the chemical cleavage method (see Cotton et al., Proc NatlAcad Sci USA (1985) 85: 4397-4401). In another embodiment, an array ofoligonucleotides probes comprising p101 nucleotide sequence or fragmentsthereof can be constructed to conduct efficient screening of e.g.,genetic mutations. Array technology methods are well known and havegeneral applicability and can be used to address a variety of questionsin molecular genetics including gene expression, genetic linkage, andgenetic variability (see for example: M.Chee et al., Science, Vol 274,pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or determining asusceptibility to the Diseases through detection of mutation in the p101gene by the methods described. In addition, such diseases may bediagnosed by methods comprising determining from a sample derived from asubject an abnormally decreased or increased level of polypeptide ormRNA. Decreased or increased expression can be measured at the RNA levelusing any of the methods well known in the art for the quantitation ofpolynucleotides, such as, for example, nucleic acid amplification, forinstance PCR, RT-PCR, RNase protection, Northern blotting and otherhybridization methods. Assay techniques that can be used to determinelevels of a protein, such as a polypeptide of the present invention, ina sample derived from a host are well-known to those of skill in theart. Such assay methods include radioimmunoassays, competitive-bindingassays, Western Blot analysis and ELISA assays.

Thus in another aspect, the present invention relates to a diagonostickit which comprises:

(a) a polynucleotide of the present invention, preferably the nucleotidesequence SEQ ID NO: 1, 3, 7 and 9 respectively, or a fragment thereof;

(b) a nucleotide sequence complementary to that of (a);

(c) a polypeptide of the present invention, preferably the polypeptideof SEQ ID NO:2, 4, 8 and 10 respectively or a fragment thereof; or

(d) an antibody to a polypeptide of the present invention, preferably tothe polypeptide of SEQ ID NO:2, 4, 8 and 10 respectively.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or suspectability to a disease, particularlydiseases that involve leucocyte activation and infiltration includinginflammatory diseases such as COPD, ARDS, arthritis, psoriasis and soon, amongst others.

The nucleotide sequences of the present invention are also valuable forchromosomal localisation. The sequence is specifically targeted to, andcan hybridize with, a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound in, for example, V. McKusick, Mendelian Inheritance in Man(available on-line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes).

The differences in the cDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

The gene of the present invention maps to human chromosome 17p12-13.1.

The nucleotide sequences of the present invention are also valuable fortissue localization. Such techniques allow the determination ofexpression patterns of the human p101 polypeptides in tissues bydetection of the mRNAs that encode them. These techniques include insitu hybridziation techniques and nucleotide amplification techniques,for example PCR. Such techniques are well known in the art. Results fromthese studies provide an indication of the normal functions of thepolypeptides in the organism. In addition, comparative studies of thenormal expression pattern of human p101 mRNAs with that of mRNAs encodedby a human p101 gene provide valuable insights into the role of mutanthuman p101 polypeptides, or that of inappropriate expression of normalhuman p101 polypeptides, in disease. Such inappropriate expression maybe of a temporal, spatial or simply quantitative nature.

The polypeptides of the invention or their fragments or analogs thereof,or cells expressing them, can also be used as immunogens to produceantibodies immunospecific for polypeptides of the present invention. Theterm “immunospecific” means that the antibodies have substantiallygreater affinity for the polypeptides of the invention than theiraffinity for other related polypeptides in the prior art.

Antibodies generated against polypeptides of the present invention maybe obtained by administering the polypeptides or epitope-bearingfragments, analogs or cells to an animal, preferably a non-human animal,using routine protocols. For preparation of monoclonal antibodies, anytechnique which provides antibodies produced by continuous cell linecultures can be used. Examples include the hybridoma technique (Kohler,G. and Milstein, C., Nature (1975) 256:495-497), the trioma technique,the human B-cell hybridoma technique (Kozbor et al., Immunology Today(1983) 4:72) and the EBV-hybridoma technique (Cole et al., MonoclonalAntibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies, such as thosedescribed in U.S. Pat. No. 4,946,778, can also be adapted to producesingle chain antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms, including other mammals, may beused to express humanized antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

Antibodies against polypeptides of the present invention may also beemployed to treat the Diseases, amongst others.

In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor x_(a). Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

Another aspect of the invention relates to a method for inducing animmunological response in a mammal which comprises inoculating themammal with a polypeptide of the present invention, adequate to produceantibody and/or T cell immune response to protect said animal from theDiseases hereinbefore mentioned, amongst others. Yet another aspect ofthe invention relates to a method of inducing immunological response ina mammal which comprises delivering a polypeptide of the presentinvention via a vector directing expression of the polynucleotide andcoding for the polypeptide in vivo in order to induce such animmunological response to produce antibody to protect said animal fromdiseases.

A further aspect of the invention relates to an immunological/vaccineformulation (composition) which, when introduced into a mammalian host,induces an immunological response in that mammal to a polypeptide of thepresent invention wherein the composition comprises a polypeptide orpolynucleotide of the present invention. The vaccine formulation mayfurther comprise a suitable carrier. Since a polypeptide may be brokendown in the stomach, it is preferably administered parenterally (forinstance, subcutaneous, intramuscular, intravenous, or intradermalinjection). Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation instonic with the blood of the recipient; and aqueous andnon-aqueous sterile suspensions which may include suspending agents orthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampoules and vials and may bestored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use. The vaccine formulationmay also include adjuvant systems for enhancing the immunogenicity ofthe formulation, such as oil-in water systems and other systems known inthe art. The dosage will depend on the specific activity of the vaccineand can be readily determined by routine experimentation.

Polypeptides of the present invention are responsible for one or morebiological functions, including one or more disease states, inparticular the Diseases hereinbefore mentioned. It is therefore desirousto devise screening methods to identify compounds which stimulate orwhich inhibit the function of the polypeptide. Accordingly, in a furtheraspect, the present invention provides for a method of screeningcompounds to identify those which stimulate or which inhibit thefunction of the polypeptide. In general, agonists or antagonists may beemployed for therapeutic and prophylactic purposes for such Diseases ashereinbefore mentioned. Compounds may be identified from a variety ofsources, for example, cells, cell-free preparations, chemical libraries,and natural product mixtures. Such agonists, antagonists or inhibitorsso-identified may be natural or modified substrates, ligands, receptors,enzymes, etc., as the case may be, of the polypeptide; or may bestructural or functional mimetics thereof (see Coligan et al., CurrentProtocols in Immunology 1(2):Chapter 5 (1991)).

The screening method may simply measure the binding of a candidatecompound to the polypeptide, or to cells or membranes bearing thepolypeptide, or a fusion protein thereof by means of a label directly orindirectly associated with the candidate compound. Alternatively, thescreening method may involve competition with a labeled competitor.Further, these screening methods may test whether the candidate compoundresults in a signal generated by activation or inhibition of thepolypeptide, using detection systems appropriate to the cells bearingthe polypeptide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Constitutivelyactive polypeptides may be employed in screening methods for inverseagonists or inhibitors, in the absence of an agonist or inhibitor, bytesting whether the candidate compound results in inhibition ofactivation of the polypeptide. Further, the screening methods may simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide of the present invention, to form a mixture,measuring p101 activity in the mixture, and comparing the p101 activityof the mixture to a standard. Fusion proteins, such as those made fromFc portion and p101 polypeptide, as hereinbefore described, can also beused for high-throughput screening assays to identify antagonists forthe polypeptide of the present invention (see D. Bennett et al., J MolRecognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

The polynucleotides, polypeptides and antibodies to the polypeptide ofthe present invention may also be used to configure screening methodsfor detecting the effect of added compounds on the production of mRNAand polypeptide in cells. For example, an ELISA assay may be constructedfor measuring secreted or cell associated levels of polypeptide usingmonoclonal and polyclonal antibodies by standard methods known in theart. This can be used to discover agents which may inhibit or enhancethe production of polypeptide (also called antagonist or agonist,respectively) from suitably manipulated cells or tissues.

The polypeptide may be used to identify membrane bound or solublereceptors, if any, through standard receptor binding techniques known inthe art. These include, but are not limited to, ligand binding andcrosslinking assays in which the polypeptide is labeled with aradioactive isotope (for instance, ¹²⁵I), chemically modified (forinstance, biotinylated), or fused to a peptide sequence suitable fordetection or purification, and incubated with a source of the putativereceptor (cells, cell membranes, cell supernatants, tissue extracts,bodily fluids). Other methods include biophysical techniques such assurface plasmon resonance and spectroscopy. These screening methods mayalso be used to identify agonists and antagonists of the polypeptidewhich compete with the binding of the polypeptide to its receptors, ifany. Standard methods for conducting such assays are well understood inthe art.

Examples of potential polypeptide antagonists include antibodies or, insome cases, oligonucleotides or proteins which are closely related tothe ligands, substrates, receptors, enzymes, etc., as the case may be,of the polypeptide, e.g., a fragment of the ligands, substrates,receptors, enzymes, etc.; or small molecules which bind to thepolypeptide of the present invention but do not elicit a response, sothat the activity of the polypeptide is prevented.

Thus, in another aspect, the present invention relates to a screeningkit for identifying agonists, antagonists, ligands, receptors,substrates, enzymes, etc. for polypeptides of the present invention; orcompounds which decrease or enhance the production of such polypeptides,which comprises:

(a) a polypeptide of the present invention;

(b) a recombinant cell expressing a polypeptide of the presentinvention;

(c) a cell membrane expressing a polypeptide of the present invention;or

(d) antibody to a polypeptide of the present invention;

which polypeptide is preferably that of SEQ ID NO:2,4, 8 and 10.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component.

It will be readily appreciated by the skilled artisan that a polypeptideof the present invention may also be used in a method for thestructure-based design of an agonist, antagonist or inhibitor of thepolypeptide, by:

(a) determining in the first instance the three-dimensional structure ofthe polypeptide;

(b) deducing the three-dimensional structure for the likely reactive orbinding site(s) of an agonist, antagonist or inhibitor;

(c) synthesing candidate compounds that are predicted to bind to orreact with the deduced binding or reactive site; and

(d) testing whether the candidate compounds are indeed agonists,antagonists or inhibitors.

It will be further appreciated that this will normally be an iterativeprocess.

In a further aspect, the present invention provides methods of treatingabnormal conditions such as, for instance, diseases that involveleucocyte activation and infiltration including inflammatory diseasessuch as COPD, ARDS, arthritis, psoriasis and so on, related to either anexcess of, or an under-expression of, p101 polypeptide activity.

If the activity of the polypeptide is in excess, several approaches areavailable. One approach comprises administering to a subject in needthereof an inhibitor compound (antagonist) as hereinabove described,optionally in combination with a pharmaceutically acceptable carrier, inan amount effective to inhibit the function of the polypeptide, such as,for example, by blocking the binding of ligands, substrates, receptors,enzymes, etc., or by inhibiting a second signal, and thereby alleviatingthe abnormal condition. In another approach, soluble forms of thepolypeptides still capable of binding the ligand, substrate, enzymes,receptors, etc. in competition with endogenous polypeptide may beadministered. Typical examples of such competitors include fragments ofthe p101 polypeptide.

In still another approach, expression of the gene encoding endogenousp101 polypeptide can be inhibited using expression blocking techniques.Known such techniques involve the use of antisense sequences, eitherinternally generated or externally administered (see, for example,O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). Alternatively, oligonucleotides which form triple helices(“triplexes”) with the gene can be supplied (see, for example, Lee etal., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al., Science (1991) 251:1360). These oligomers can beadministeredper se or the relevant oligomers can be expressed in vivo.Synthetic antisense or triplex oligonucleotides may comprise modifiedbases or modified backbones. Examples of the latter includemethylphosphonate, phosphorothioate or peptide nucleic acid backbones.Such backbones are incorporated in the antisense or triplexoligonucleotide in order to provide protection from degradation bynucleases and are well known in the art. Antisense and triplex moleculessynthesized with these or other modified backbones also form part of thepresent invention.

In addition, expression of the human p101 polypeptide may be preventedby using ribozymes specific to the human p101 mRNA sequence. Ribozymesare catalytically active RNAs that can be natural or synthetic (see forexample Usman, N, et al., Curr. Opin. Struct. Biol (1996) 6(4), 527-33.)Synthetic ribozymes can be designed to specifically cleave human p101mRNAs at selected positions thereby preventing translation of the humanp101 mRNAs into a functional polypeptide. Ribozymes may be synthesisedwith a natural ribose phosphate backbone and natural bases, as normallyfound in RNA molecules. Alternatively the ribozymes may be synthesizedwith non-natural backbones to provide protection from ribonucleasedegradation, for example, 2′-O-methyl RNA, and may contain modifiedbases.

For treating abnormal conditions related to an under-expression of p101and its activity, several approaches are also available. One approachcomprises administering to a subject a therapeutically effective amountof a compound which activates a polypeptide of the present invention,i.e., an agonist as described above, in combination with apharmaceutically acceptable carrier, to thereby alleviate the abnormalcondition. Alternatively, gene therapy may be employed to effect theendogenous production of p101 by the relevant cells in the subject. Forexample, a polynucleotide of the invention may be engineered forexpression in a replication defective retroviral vector, as discussedabove. The retroviral expression construct may then be isolated andintroduced into a packaging cell transduced with a retroviral plasmidvector containing RNA encoding a polypeptide of the present inventionsuch that the packaging cell now produces infectious viral particlescontaining the gene of interest. These producer cells may beadministered to a subject for engineering cells in vivo and expressionof the polypeptide in vivo. For an overview of gene therapy, see Chapter20, Gene Therapy and other Molecular Genetic-based TherapeuticApproaches, (and references cited therein) in Human Molecular Genetics,T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996). Anotherapproach is to administer a therapeutic amount of a polypeptide of thepresent invention in combination with a suitable pharmaceutical carrier.

In a further aspect, the present invention provides for pharmaceuticalcompositions comprising a therapeutically effective amount of apolypeptide, such as the soluble form of a polypeptide of the presentinvention, agonist/antagonist peptide or small molecule compound, incombination with a pharmaceutically acceptable carrier or excipient.Such carriers include, but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theinvention further relates to pharmaceutical packs and kits comprisingone or more containers filled with one or more of the ingredients of theaforementioned compositions of the invention. Polypeptides and othercompounds of the present invention may be employed alone or inconjunction with other compounds, such as therapeutic compounds.

The composition will be adapted to the route of administration, forinstance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, and the like.

The dosage range required depends on the choice of peptide or othercompounds of the present invention, the route of administration, thenature of the formulation, the nature of the subject's condition, andthe judgment of the attending practitioner. Suitable dosages, however,are in the range of 0.1-100 μg/kg of subject. Wide variations in theneeded dosage, however, are to be expected in view of the variety ofcompounds available and the differing efficiencies of various routes ofadministration. For example, oral administration would be expected torequire higher dosages than administration by intravenous injection.Variations in these dosage levels can be adjusted using standardempirical routines for optimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in thesubject, in treatment modalities often referred to as “gene therapy” asdescribed above. Thus, for example, cells from a subject may beengineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

Polynucleotide and polypeptide sequences form a valuable informationresource with which to identify further sequences of similar homology.This is most easily facilitated by storing the sequence in a computerreadable medium and then using the stored data to search a sequencedatabase using well known searching tools, such as those in the GCG orLasergene software packages. Accordingly, in a further aspect, thepresent invention provides for a computer readable medium having storedthereon a polynucleotide comprising the sequence of SEQ ID NO: 1 and/ora polypeptide sequence encoded thereby.

The following definitions are provided to facilitate understanding ofcertain terms used frequently hereinbefore.

“Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

“Isolated” means altered “by the hand of man” from the natural state. Ifan “isolated” composition or substance occurs in nature, it has beenchanged or removed from its original environment, or both. For example,a polynucleotide or a polypeptide naturally present in a living animalis not “isolated,” but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is “isolated”, as theterm is employed herein.

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term “polynucleotide” also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

“Polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications may occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentto the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from post-translation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, biotinylation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination (see, for instance,Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York, 1993; Wold, F., Post-translationalProtein Modifications: Perspectives and Prospects, pgs. 1-12 inPost-translational Covalent Modification of Proteins, B. C. Johnson,Ed., Academic Press, New York, 1983; Seifter et al., “Analysis forprotein modifications and nonprotein cofactors”, Meth Enzymol (1990)182:626-646 and Rattan et al., “Protein Synthesis: Post-translationalModifications and Aging”, Ann NY Acad Sci (1992) 663:48-62).

“Variant” refers to a polynucleotide or polypeptide that differs from areference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48. 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1).387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec.Biol. 215. 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

Preferred parameters for polypeptide sequence comparison include thefollowing:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Preferred parameters for polynucleotide comparison include thefollowing:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The “gap” program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

By way of example, a polynucleotide sequence of the present inventionmay be identical to the reference sequence of SEQ ID NO:1, that is be100% identical, or it may include up to a certain integer number ofnucleotide alterations as compared to the reference sequence. Suchalterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleotide alterations is determined by multiplying the total number ofnucleotides in SEQ ID NO: 1 by the numerical percent of the respectivepercent identity(divided by 100) and subtracting that product from saidtotal number of nucleotides in SEQ ID NO: 1, or:

n _(n) ≦x _(n)−(x _(n) ·y)

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO: 1, and y is, for instance,0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%,etc., and wherein any non-integer product of x_(n) and y is roundeddown to the nearest integer prior to subtracting it from x_(n).Alterations of a polynucleotide sequence encoding the polypeptide of SEQID NO:2 may create nonsense, missense or frameshift mutations in thiscoding sequence and thereby alter the polypeptide encoded by thepolynucleotide following such alterations.

Similarly, a polypeptide sequence of the present invention may beidentical to the reference sequence of SEQ ID NO:2, that is be 100%identical, or it may include up to a certain integer number of aminoacid alterations as compared to the reference sequence such that the %identity is less than 100%. Such alterations are selected from the groupconsisting of at least one amino acid deletion, substitution, includingconservative and non-conservative substitution, or insertion, andwherein said alterations may occur at the amino- or carboxy-terminalpositions of the reference polypeptide sequence or anywhere betweenthose terminal positions, interspersed either individually among theamino acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of amino acidalterations for a given % identity is determined by multiplying thetotal number of amino acids in SEQ ID NO:2 by the numerical percent ofthe respective percent identity(divided by 100) and then subtractingthat product from said total number of amino acids in SEQ ID NO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y)

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, and y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein any non-integerproduct of x_(a) and y is rounded down to the nearest integer prior tosubtracting it from x_(a).

“Homolog” is a generic term used in the art to indicate a polynucleotideor polypeptide sequence possessing a high degree of sequence relatednessto a subject sequence. Such relatedness may be quantified by determiningthe degree of identity and/or similarity between the sequences beingcompared as hereinbefore described. Falling within this generic term arethe terms “ortholog”, meaning a polynucleotide or polypeptide that isthe functional equivalent of a polynucleotide or polypeptide in anotherspecies, and “paralog” meaning a functionally similar sequence whenconsidered within the same species.

“Fusion protein” refers to a protein encoded by two, often unrelated,fused genes or fragments thereof. In one example, EP-A-0 464 disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, employing an immunoglobulin Fc region as a partof a fusion protein is advantageous for use in therapy and diagnosisresulting in, for example, improved pharmacokinetic properties [see,e.g., EP-A 0232 262]. On the other hand, for some uses it would bedesirable to be able to delete the Fc part after the fusion protein hasbeen expressed, detected and purified.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

Sequence Information

(SVP-2,lacks exons 6,7,8,9 and 10) SEQ ID NO:1CCCTTTCCACCTCTCTGCTCCCATTCCTGACCCCTTACTTCCCACACCTCTGTCCCGTTCTGCTGCAGGGGTGCTCTGTCCTGCCACTCAGATGTGGCCCTCCACATGCCATTCCTACCCTGGAGGCAGCTGTAAGGCCCCTGGTCCTGTTTCCACAGCACCTGAGCTATAGCTGGGCTGGGCTGATCGCGCTGCACTGTGAGCACCTGTTGTCTTTACTGGACCAGGTGCTCTCTGGGAAAGGAGCTCGACAAGCTGACCGGCGTCTGTCCCCCATGCAGGCGATGACCCAGGATGCAGCCAGGGGCCACGACATGCACGGAGGACCGCATCCAGCATGCCCTGGAACGCTGCCTGCATGGACTCAGCCTCAGCCGCCGCTCCACCTCCTGGTCAGCTGGGCTGTGTCTGAACTGCTGGAGCCTGCAGGAGCTGGTCAGCAGGGACCCGGGCCACTTCCTTATCCTCCTTGAGCAGATCCTGCAGAAGACCCGAGAGGTCCAGGAGAAGGGCACCTACGACCTGCTCACCCCGCTGGCCCTGCTCTTCTATTCCACTGTTCTTTGTACACCACACTTCCCACCAGACTCGGATCTCCTTCTGAAGGCAGCCAGCACCTACCACCGGTTCCTGACCTGGCCTGTTCCTTACTGCAGCATCTGCCAGGAGCTGCTCACCTTCATTGATGCTGAACTCAAGGCCCCAGGGATCTCCTACCAGAGACTGGTGAGGGCTGAGCAGGGCCTGCCCATCAGGAGTCACCGCAGCTCCACCGAGCTGGGCACCACCCCATGGGAGGAGAGCACCAATGGCATCTCCCACTACCTCGGCATGCTGGACCCCTGGTATGAGCGCAATGTACTGGGCCTCATGCACCTGCCCCCTGAAGTCCTGTGCCAGCAGTCCCTGAAGGCTGAAGCCCAGGCCCTGGAGGGCTCCCCAACCCAGCTGCCCATCCTGGCTGACATGCTACTCTACTACTGCCGCTTTGCCGCCAGACCGGTGCTGCTGCAAGTCTATCAGACCGAGCTGACCTTCATCACTGGGGAGAAGACGACAGAGATCTTCATCCACTCCTTGGAGCTGGGTCACTCCGCTGCCACACGTGCCATCAAGGCGTCAGGTCCTGGCAGCAAGCGGCTGGGCATCGATGGCGACCGGGAGGCTGTTCCTCTAACACTACAGATTATTTACAGCCAGGGGGCCATCAGTGGACGAAGTCGCTGGAGCAACCTGGAGAAGGTCTGTACCTCCGTGAACCTCAACAAGGCCTGCCGGAAGCAGGAGGAGCTGGATTCCAGCATGGAGGCCCTGACGCTAAACCTGACAGAAGTGGTGAAAAGGCAGAACTCCAAATCCAAGAAGGGCTTTAACCAGATTAGCACATCGCAGATCAAAGTGGACAAGGTGCAGATCATCGGCTCCAACAGCTGCCCCTTTGCTGTGTGCCTCCACCAGGATGAGAGAAAGATCCTGCAGAGTGTAGTCAGATGTGAGGTCTCACCGTGCTACAAGCCAGAGAAGAGCGACCTCTCCTCACCACCCCAGACGCCTCCTGACCTGCCGGCCCAGGCCGCACCTGATCTCTGCTCCCTCCTCTGCCTGCCCATCATGACTTTCAGTGGAGCTCTGCCCTAGTGTGGGCCCAGCGCCACACTGGACAGAAGCCCTGGGGTCATTTCTCCAGCACTAAAATGGAGTGGAGAGTTGGGGTGGAAATAAGACATCCTTAAAAGGTTAAATTGTCTGCAAAGCACCTAGCCCAGTGCCGAGCTCCCAGTAGGTGTTCAGTAAAGCTTAGTGCCTGACTTTCTGAACACTGATTCCTCCTGTTTGGAGTCACTGGGATACTCTCATTGCCGTTGGGATGTTCCTCACTCCTTCCCAGTTCGTGGCTGAGGCAGAACCCAGACTGAAGAGGGAAGAGACATTCCAGAGGAGGATTGCCTTCGTCAGGGTAAGGGGTGGGCTGCTCAGGGGCCCTACCCTTCACCCCCTTCTGTATCAGATTGGCCCTCCCACTCCCATCTCACTCTGCGTGTACAATCTTCCATATCCGCAAGTTCACTGGCACTCTTCTGGCACCTGGGCAAGATCCCAGAACAGAGGATGGAGTGACTGGCCTCACAGAGCTTAGTGCCCGACACTGGTGCATGGGAAATGGTCAGCCTAGGATAGGACACGAGAGTCTGAAATTCAAAGCAACCAGCTTGAAGTGGTTTGAGAAGCTGGAAGCAAACATGGGCTAGAGAGATAGGGCAGAAGTCAAGACGAGGATCTGGACTGATGTGGAGAAAGTAGCCACGGAAGCATGAACTGTATCCTGCACAAAGTCCCTCTTCCCCGCCTCCTAATTCATTATGCCCAAAAGGCCTTACGTGAAATTCCAGCCCACACTACTCATGACTTGAGAGACGTGGACAGAGCCAGCTTCTACCTTGCCTGGCCGTCTCTCCCCTGTCTTAATGTCTGCTCTTGCTCTAAGCTCCAGAAGAGTGGCGGGCCATGTATCTTCAATATGTTTTTGCTGTATGGGCAGGTTGTCTTATTATGTGATCAACAGATGTCCAGGAACTAATGAGTGGAATTTAATATTATTGTCAAATAAAACTTGATTTGTCCTAT (SVP-2 protein) SEQ ID NO:2MQOGATTCTEDRIQHALERCLHGLSLSRRSTSWSAGLCLNCWSLQELVSRDPGHFLILLEQILQKTREVQEKGTYDLLTPLALLFYSTVLCTPHFPPDSDLLLKAASTYHRFLTWPVPYCSICQELLTFIDAELKAPGISYQRLVRAEQGLPIRSHRSSTELGTTPWEESTNGISHYLGMLDPWYERNVLGLMHLPPEVLCQQSLKAEAQALEGSPTQLPILADMLLYYCRFAARPVLLQVYQTELTFITGEKTTEIFIHSLELGHSAATRAIKASGPGSKRLGIDGDREAVPLTLQIIYSQGAISGRSRWSNLEKVCTSVNLNKACRKQEELDSSMEALTLNLTEVVKRQNSKSKKGFNQISTSQIKVDKVQIIGSNSCPFAVCLDQDERKILQSVVRCEVSPCYKPEKSDLSSPPQTPPDLPAQAAPDLCSLLCLPIMTFSGALP (SVP-4, lacks exons 9 and 10) SEQ ID NO:3CCCTTTCCACCTCTCTGCTCCCATTCCTGACCCCTTACTTCCCACACCTCTGTCCCGTTCTGCTGCAGGGGTGCTCTGTCCTGCCACTCAGATGTGGCCCTCCACATGCCATTCCTACCCTGGAGGCAGCTGTAAGGCCCCTGGTCCTGTTTCCACAGCACCTGAGCTATAGCTGGGCTGGGCTGATCGCGCTGCACTGTGAGCACCTGTTGTCTTTACTGGACCAGGTGCTCTCTGGGAAAGGAGCTCGACAAGCTGACCGGCGTCTGTCCCCCATGCAGGCGATGACCCAGGATGCAGCCAGGGGCCACGACATGCACGGAGGACCGCATCCAGCATGCCCTGGAACGCTGCCTGCATGGACTCAGCCTCAGCCGCCGCTCCACCTCCTGGTCAGCTGGGCTGTGTCTGAACTGCTGGAGCCTGCAGGAGCTGGTCAGCAGGGACCCGGGCCACTTCCTTATCCTCCTTGAGCAGATCCTGCAGAAGACCCGAGAGGTCCAGGAGAAGGGCACCTACGACCTGCTCACCCCGCTGGCCCTGCTCTTCTATTCCACTGTTCTTTGTACACCACACTTCCCACCAGACTCGGATCTCCTTCTGAAGGCAGCCAGCACCTACCACCGGTTCCTGACCTGGCCTGTTCCTTACTGCAGCATCTGCCAGGAGCTGCTCACCTTCATTGATGCTGAACTCAAGGCCCCAGGTATCTCCTACCAGAGACTGGTGAGGGCTGAGCAGGGCCTGCCCATCAGGAGTCACCGCAGCTCCACCGTCACCGTGCTGCTGCTGAACCCAGTGGAAGTGCAGGCCGAGTTCCTTGCTGTAGCCAATAAGCTGAGTACGCCCGGACACTCGCCTCACAGTGCCTACACCACCCTGCTCCTGCACGCCTTCCAGGCCACCTTTGGGGCCCACTGTGACGTCCCGGGCCTGCACTGCAGGCTACAGGCCAAGACCCTGGCAGAGCTTGAGGACATCTTCACGGAGACCGCAGAGGCACAGGAGCTGGCATCTGGCATCGGGGATGCTGCAGAGGCCCGGCGGTGGCTCAGGACCAAGCTGCAGGCGGTGGGAGAAAAAGCTGGCTTCCCTGGGGTGTTAGACACTGCAAAACCAGGGAAGCTTCATACCATCCCCATCCCTGTCGCCAGGTGCTACACCTACAGCTGGAGCCAGGACAGCTTTGGAGCTGGGCACCACCCCATGGGAGGAGAGCACCAATGGCATCTCCCACTACCTCGGCATGCTGGACCCCTGGTATGAGCGCAATGTACTGGGCCTCATGCACCTGCCCCCTGAAGTCCTGTGCCAGCAGTCCCTGAAGGCTGAAGCCCAGGCCCTGGAGGGCTCCCCAACCCAGCTGCCCATCCTGGCTGACATGCTACTCTACTACTGCCGCTTTGCCGCCAGACCGGTGCTGCTGCAAGTCTATCAGACCGAGCTGACCTTCATCACTGGGGAGAAGACGACAGAGATCTTCATCCACTCCTTGGAGCTGGGTCACTCCGCTGCCACACGTGCCATCAAGGCGTCAGGTCCTGGCAGCAAGCGGCTGGGCATCGATGGCGACCGGGAGGCTGTTCCTCTAACACTACAGATTATTTACAGCCAGGGGGCCATCAGTGGACGAAGTCGCTGGAGCAACCTGGAGAAGGTCTGTACCTCCGTGAACCTCAACAAGGCCTGCCGGAAGCAGGAGGAGCTGGATTCCAGCATGGAGGCCCTGACGCTAAACCTGACAGAAGTGGTGAAAAGGCAGAACTCCAAATCCAAGAAGGGCTTTAACCAGATTAGCACATCGCAGATCAAAGTGGACAAGGTGCAGATCATCGGCTCCAACAGCTGCCCCTTTGCTGTGTGCCTGGACCAGGATGAGAGAAAGATCCTGCAGAGTGTAGTCAGATGTGAGGTCTCACCGTGCTACAAGCCAGAGAAGAGCGACCTCTCCTCACCACCCCAGACGCCTCCTGACCTGCCGGCCCAGGCCGCACCTGATCTCTGCTCCCTCCTCTGCCTGCCCATCATGACTTTCAGTGGAGCTCTGCCCTAGTGTGGGCCCAGCGCCAGACTGGACAGAAGCCCTGGGGTCATTTCTCCAGCACTAAAATGGAGTGGAGAGTTGGGGTGGAAATAAGACATCCTTAAAAGGTTAAATTGTCTGCAAAGCACCTAGCCCAGTGCCGAGCTCCCAGTAGGTGTTCAGTAAAGCTTAGTGCCTGACTTTCTGAACACTGATTCCTCCTGTTTGGAGTCACTGGGATACTCTCATTGCCGTTGGGATGTTCCTCACTCCTTCCCAGTTCGTGGCTGAGGCAGAACCCAGACTGAAGAGGGAAGAGACATTCCAGAGGAGGATTGCCTTCGTCAGGGTAAGGGGTGGGCTGCTCAGGGGCCCTACCCTTCACCCCCTTCTGTATCAGATTGGCCCTCCCACTCCCATCTCACTCTGCGTGTACAATCTTCCATATCCGCAAGTTCACTGGCACTCTTCTGGCACCTGGGCAAGATCCCAGAACAGAGGATGGAGTGACTGGCCTCACAGAGCTTAGTGCCCGACACTGGTGCATGGGAAATGGTCAGCCTAGGATAGGACACGAGAGTCTGAAATTCAAAGCAACCAGCTTGAAGTGGTTTGAGAAGCTGGAAGCAAACATGGGCTAGAGAGATAGGGCAGAAGTCAAGACGAGGATGTGGACTGATGTGGAGAAAGTAGCCACGGAAGCATGAACTGTATCCTGCACAAAGTCCCTCTTCCCCGCCTCCTAATTCATTATGCCCAAAAGGCCTTACGTGAAATTCCAGCCCAGAGTACTCATGACTTGAGAGACGTGGACAGAGCCAGCTTCTACCTTGCCTGGCCGTCTCTCCCCTGTCTTAATGTCTGCTCTTGCTCTAAGCTCCAGAAGAGTGGCGGGCCATGTATCTTCAATATGTTTTTGCTGTATGGGCAGGTTGTCTTATTATGTGATCAACAGATGTCCAGGAACTAATGAGTGGAATTTAATATTATTGTCAAATAAAACTTGATTTGTCCTAT (SVP-4 protein) SEQ ID NO:4MQPGATTCTEDRIQHALERCLHGLSLSRRSTSWSAGLCLNCWSLQELVSRDPGHFLILLEQILQKTREVQEKGTYDLLTPLALLFYSTVLCTPHFPPDSDLLLKAASTYHRFLTWPVPYCSISQWLLTFIDAELKAPGISYQRLVRAEQGLPIRSHRSSTVTVLLLNPVEVQAEFLAVANKLSTPGHSPHSAYTTLLLHAFQATFGAHCDVPGLHCRLQAKTLAELEDIFTETAEAQELASGIGDAAEARRWLRTKLQAVGEKAGFPGVLDTAKPGKLHTIPIPVARCYTYSWSQDSFGAGHHPMGGEHQWHLPLPRHAGPLV (Full-length human p101 DNA) SEQ IDNO:5ATGCAGCCAGGGGCCACGACATGCACGGAGGACCGCATCCAGCATGCCCTGGAACGCTGCCTGCATGGACTCAGCCTCAGCCGCCGCTCCACCTCCTGGTCAGCTGGGCTGTGTCTGAACTGCTGGAGCCTGCAGGAGCTGGTCAGCAGGGACCCGGGCCACTTCCTTATCCTCCTTGAGCAGATCCTGCAGAAGACCCGAGAGGTCCAGGAGAAGGGCACCTACGACCTGCTCACCCCGCTGGCCCTGCTCTTCTATTCCACTGTTCTTTGTACACCACACTTCCCACCAGACTCGGATCTCCTTCTGAAGGCAGCCAGCACCTACCACCGGTTCCTGACCTGGCCTGTTCCTTACTGCAGCATCTGCCAGGAGCTGCTCACCTTCATTGATGCTGAACTCAAGGCCCCAGGGATCTCCTACCAGAGACTGGTGAGGGCTGAGCAGGGCCTGCCCATCAGGAGTCACCGCAGCTCCACCGTCACCGTGCTGCTGCTGAACCCAGTGGAAGTGCAGGCCGAGTTCCTTGCTGTAGCCAATAAGCTGAGTACGCCCGGACACTCGCCTCACAGTGCCTACACCACCCTGCTCCTGCACGCCTTCCAGGCCACCTTTGGGGCCCACTGTGACGTCCCGGGCCTGCACTGCAGGCTACAGGCCAAGACCCTGGCAGAGCTTGAGGACATCTTCACGGAGACCGCAGAGGCACAGGAGCTGGCATCTGGCATCGGGGATGCTGCAGAGGCCCGGCGGTGGCTCAGGACCAAGCTGCAGGCGGTGGGAGAAAAAGCTGGCTTCCCTGGGGTGTTAGACACTGCAAAACCAGGGAAGCTTCATACCATCCCCATCCCTGTCGCCAGGTGCTACACCTACAGCTGGAGCCAGGACAGCTTTGACATCCTGCAGGAAATCCTGCTCAAGGAACAGGAGCTACTCCAGCCAGGGATCCTGGGAGATGATGAAGAGGAGGAAGAGGAGGAGGAGGAGGTGGAGGAGGACTTGGAAACTGACGGGCACTGTGCCGAGAGAGATTCCCTGCTCTCCACCAGCTCTTTGGCGTCCCATGACTCCACCTTGTCCCTTGCATCCTCCCAGGCCTCGGGGCCGGCCCTCTCGCGCCATCTGCTGACTTCCTTTGTCTCAGGCCTCTCTGATGGCATGGACAGCGGCTACGTGGAGGACAGCGAGGAGAGCTCCTCCGAGTGGCCTTGGAGGCGTGGCAGCCAGGAACGCCGAGGCCACCGCAGGCCTGGGCAGAAGTTCATCAGGATCTATAAACTCTTCAAGAGCACCAGCCAGCTGGTACTGCGGAGGGACTCTCGGAGCCTGGAGGGCAGCTCGGACACGGCCCTGCCCCTGAGGCGGGCAGGGAGCCTCTGCAGCCCCCTGGACGAACCAGTATCACCCCCTTCCCGGGCCCAGCGCTCCCGCTCCCTGCCCCAGCCCAAACTCGGTACCCAGCTGCCCAGCTGGCTTCTGGCCCCTGCTTCACGCCCCCAGCGCCGCCGCCCCTTCCTGAGTGGAGATGAGGATCCCAAGGCTTCCACGCTACGTGTTGTGGTCTTTGGCTCCGATCGGATTTCAGGGAAGGTGGCTCGGGCGTACAGCAACCTTCGGCGGCTGGAGAACAATCGCCCACTCCTCACACGGTTCTTCAAACTTCAGTTCTTCTACGTGCCTGTGAAGCGAAGTCGTGGGACCAGCCCTGGTGCCTGTCCACCCCCTCGGAGCCAGACGCCCTCACCCCCGACAGACTCCCCTAGGCACGCCAGCCCTGGAGAGCTGGGCACCACCCCATGGGAGGAGAGCACCAATGGCATCTCCCACTACCTCGGCATGCTGGACCCCTGGTATGAGCGCAATGTACTGGGCCTCATGCACCTGCCCCCTGAAGTCCTGTGCCAGCAGTCCCTGAAGGCTGAAGCCCAGGCCCTGGAGGGCTCCCCAACCCAGCTGCCCATCCTGGCTGACATGCTACTCTACTACTGCCGCTTTGCCGCCAGACCGGTGCTGCTGCAAGTCTATCAGACCGAGCTGACCTTCATCACTGGGGAGAAGACGACAGAGATCTTCATCCACTCCTTGGAGCTGGGTCACTCCGCTGCCACACGTGCCATCAAGGCGTCAGGTCCTGGCAGCAAGCGGCTGGGCATCGATGGCGACCGGGAGGCTGTTCCTCTAACACTACAGATTATTTACAGCCAGGGGGCCATCAGTGGACGAAGTCGCTGGAGCAACCTGGAGAAGGTCTGTACCTCCGTGAACCTCAACAAGGCCTGCCGGAAGCAGGAGGAGCTGGATTCCAGCATGGAGGCCCTGACGCTAAACCTGACAGAAGTGGTGAAAAGGCAGAACTCCAAATCCAAGAAGGGCTTTAACCAGATTAGCACATCGCAGATCAAAGTGGACAAGGTGCAGATCATCGGCTCCAACAGCTGCCCCTTTGCTGTGTGCCTGGACCAGGATGAGAGAAAGATCCTGCAGAGTGTAGTCAGATGTGAGGTCTCACCGTGCTACAAGCCAGAGAAGAGCGACCTCTCCTCACCACCCCAGACGCCTCCTGACCTGCCGGCCCAGGCCGCACCTGATCTCTGCTCCCTCCTCTGCCTGCCCATCATGACTTTCAGTGGAGCTCTGCCCTAGTGTGGGCCCAGCGCCACACTGGACAGAAGCCCTGGGGTCATTTCTCCAGCACTAAAATGGAGTGGAGAGTTGGGGTGGAAATAAGACATCCTTAAAAGGTTAAATTGTCTGCAAAGCACCTAGCCCAGTGCCGAGCTCCCAGTAGGTGTTCAGTAAAGCTTAGTGCCTGACTTTCTGAACACTGATTCCTCCTGTTTGGAGTCACTGGGATACTCTCATTGCCGTTGGGATGTTCCTCACTCCTTCCCAGTTCGTGGCTGAGGCAGAACCCAGACTGAAGAGGGAAGAGACATTCCAGAGGAGGATTGCCTTCGTCAGGGTAAGGGGTGGGCTGCTCAGGGGCCCTACCCTTCACCCCCTTCTGTATCAGATTGGCCCTCCCACTCCCATCTCACTCTGCGTGTACAATCTTCCATATCCGCAAGTTCACTGGCACTCTTCTGGCACCTGGGCAAGATCCCAGAACAGAGGATGGAGTGACTGGCCTCACAGAGCTTAGTGCCCGACACTGGTGCATGGGAAATGGTCAGCCTAGGATAGGACACGAGAGTCTGAAATTCAAAGCAACCAGCTTGAAGTGGTTTGAGAAGCTGGAAGCAAACATGGGCTAGAGAGATAGGGCAGAAGTCAAGACGAGGATCTGGACTGATGTGGAGAAAGTAGCCACGGAAGCATGAACTGTATCCTGCACAAAGTCCCTCTTCCCCGCCTCCTAATTCATTATGCCCAAAAGGCCTTACGTGAAATTCCAGCCCAGAGTACTCATGACTTGAGAGACGTGGACAGAGCCAGCTTCTACCTTGCCTGGCCGTCTCTCCCCTGTCTTAATGTCTGCTCTTGCTCTAAGCTCCAGAAGAGTGGCGGGCCATGTATCTTCAATATGTTTTTGCTGTATGGGCAGGTTGTCTTATTATGTGATCAACAGATGTCCAGGAACTAATGAGTGGAATTTAATATTATTGTCAAATAAAACTTGATTTGTCCTAT (Full-length human p101 protein) SEQ IDNO:6MQPGATTCTEDRIQHALERCLHGLSLSRRSTSWSAGLCLNCWSLQELVSRDPGHFLILLEQILQKTREVQEKGTYDLLTPLALLFYSTVLCTPHFPPDSDLLLKAASTYHRFLTWPVPYCSICQELLTFIDAELKAPGISYQRLVRAEQGLPIRSHRSSTVTVLLLNPVEVQAEFLAVANKLSTPGHSPHSAYTTLLLHAFQATFGAHCDVPGLHCRLQAKTLAELEDIFTETAEAQELASGIGDAAEARRWLRTKLQAVGEKAGFPGVLDTAKPGKLHTIPIPVARCYTYSWSQDSFDILQEILLKEQELLQPGILGDDEEEEEEEEEVEEDLETDGHCAERDSLLSTSSLASHDSTLSLASSQASGPALSRHLLTSFVSGLSDGMDSGYVEDSEEDDDEWPWRRGSQERRGHRRPGQKFIRIYKLFKSTSQLVLRRDSRSLEGSSDTALPLRRAGSLCSPLDEPVSPPSRAQRSRSLPQPKLGTQLPSWLLAPASRPQRRRPFLSGDEDPKASTLRVVVFGSDRISGKVARAYSNLRRLENNRPLLTRFFKLQFFYVPVKRSRGTSPGACPPPRPTQLPILADMLLYYCRFAARPVLLQVYQTELTFEITGEKTTEFIHSLELGHSAATRAIKASGPGSKRLGIDGDREAVPLTLQIIYSQGAISGRSRWSNLEKVCTSVNLNKACRKQEELDSSMEALTLNLTEVVKRQNSKSKKGFNQISTSQIKVDKVQIIGSNSCPFAVCLDQDERKILQSVVRCEVSPCYKPEKSDLSSPPQTPPDLPAQAAPDLCSLLCLPIMTFSGALP SEQ ID NO:7CAGGCGATGACCCAGGATGCAGCCAGGGGCCACGACATGCACGGAGGACCGCATCCAGCATGCCCTGGAACGCTGCCTCGATGGACTCAGCCTCAGCCGCCGCTCCACCTCCTGGTCAGCTGGGCTGTGTCTGAACTGCTGGAGCCTGCAGGAGCTGGTCAGCAGGGACCCGGGCCACTTCCTTATCCTCCTTGAGCAGATCCTGCAGAAGACCCGAGAGGTCCAGGAGAAGGGCACCTACGACCTGCTCACCCCGCTGGCCCTGCTCTTCTATTCCACTGTTCTTTGTACACCACACTTCCCACCAGACTCGGATCTCCTTCTGAAGGCAGCCAGCACCTACCACCGGTTCCTGACCTGGCCTGTTCCTTACTGCAGCATCTGCCAGGAGCTGCTCACCTTCATTGATGCTGAACTCAAGGCCCCAGGTATCTCCTACCAGAGACTGGTGAGGGCTGAGCAGGGCCTGCCCATCAGGAGTCACCGCAGCTCCACCAGGCCTGTCCACCCCCTCGGAGCCAGACGCCCTCACCCCCGACAGACTCCCCTAGGCACGCCAGCCCTGGAGAGCTGGGCACCACCCCATGGGAGGAGAGCACCAATGGCATCTCCCACTACCTCGGCATGCTGGACCCCTGGTATGAGCGCAATGTACTGGGCCTCATGCACCTGCCCCCTGAAGTCCTGTGCCAGCAG SEQ ID NO:8MQPGATTCTEDRIQHALERCLHGLSLSRRSTSWSAGLCLNCWSLQELVSRDPGHFLILLEQILQKTREVQEKGTYDLLTPLALLFYSTVLCTPHFPPDSDLLLKAASTYHRFLTWPVPYCSICQELLTFIDAELKAPGISYQRLVRAEQGLPIRSHRSSTRPVHPLGARRPHPRQTPLGTPALESWAPPHGRRAPMASPTTSACWTPGMSAMYWASCTCPLKSCAS SEQ ID NO:9ACACCACACTTCCCACCAGACTCGGATCTCCTTCTGAAGGCAGCCAGCACCTACCACCGGTTCCTGACCTGGCCTGTTCCTTACTGCAGCATCTGCCAGGAGCTGCTCACCTTCATTGATGCTGAACTCAAGGCCCCAGGGATCTCCTACCAGAGACTGGTGAGGGCTGAGCAGGGCCTGCCCATCAGGAGTCACCGCAGCTCCACCGTCACCGTGCTGCTGCTGAACCCAGTGGAAGTGCAGGCCGAGTTCCTTGCTGTAGCCAATAAGCTGAGTACGCCCGGACACTCGCCTCACAGTGCCTACACCACCCTGCTCCTGCACGCCTTCCAGGCCACCTTTGGGGCCCACTGTGACGTCCCGGGCCTGCACTGCAGGCTACAGGCCAAGACCCTGGCAGAGCTTGAGGACATCTTCACGGAGACCGCAGAGGCACAGGAGCTGGCATCTGGCATCGGGGATGCTGCAGAGGCCCGGCGGTGGCTCAGGACCAAGCTGCAGGCGGTGGGAGAAAAAGCTGGCTTCCCTGGGGTGTTAGACACTGCAAAACCAGGGAAGCTTCATACCATCCCCATCCCTGTCGCCAGGTGCTACACCTACAGCTGGAGCCAGGACAGCTTTGGGAGCTGGGCACCACCCCATGGGAGGAGAGCACCAATGGCATCTCCCACTACCTCGGCATGCTGGACCCCTGGTATGAGCGCAATGTACTGGGCCTCATGCACCTGCCCCCTGAAGTCCTGTGCCAGCAG SEQ ID NO:10TPHFPPDSDLLLKAASTYHRFLTWPVPYCSICQELLTFIDAELKAPGISYQRLVRAEQGLPIRSHRSSTVTVLLLNOVEVQAEFLAVANKLSTPGHSPHSAYTTLLLHAFQATFGAHCDVPGLHCRLQAKTLAELEDIFTETAEAQELASGIGDAAEARRWLRTKLQAVGEKAGFPGVLDTAKPGKLHTOPIPVARCYTYSWSQDSFGSWAPPHGRRAPMASPTTSACWTPGMSAMYWASCTCPLKSCAS

10 1 2625 DNA HOMO SAPIENS 1 ccctttccac ctctctgctc ccattcctga ccccttacttcccacacctc tgtcccgttc 60 tgctgcaggg gtgctctgtc ctgccactca gatgtggccctccagatgcc attcctaccc 120 tggaggcagc tgtaaggccc ctggtcctgt ttccacagcacctgagctat agctgggctg 180 ggctgatcgc gctgcactgt gagcacctgt tgtctttactggaccaggtg ctctctggga 240 aaggagctcg acaagctgac cggcgtctgt cccccatgcaggcgatgacc caggatgcag 300 ccaggggcca cgacatgcac ggaggaccgc atccagcatgccctggaacg ctgcctgcat 360 ggactcagcc tcagccgccg ctccacctcc tggtcagctgggctgtgtct gaactgctgg 420 agcctgcagg agctggtcag cagggacccg ggccacttccttatcctcct tgagcagatc 480 ctgcagaaga cccgagaggt ccaggagaag ggcacctacgacctgctcac cccgctggcc 540 ctgctcttct attccactgt tctttgtaca ccacacttcccaccagactc ggatctcctt 600 ctgaaggcag ccagcaccta ccaccggttc ctgacctggcctgttcctta ctgcagcatc 660 tgccaggagc tgctcacctt cattgatgct gaactcaaggccccagggat ctcctaccag 720 agactggtga gggctgagca gggcctgccc atcaggagtcaccgcagctc caccgagctg 780 ggcaccaccc catgggagga gagcaccaat ggcatctcccactacctcgg catgctggac 840 ccctggtatg agcgcaatgt actgggcctc atgcacctgccccctgaagt cctgtgccag 900 cagtccctga aggctgaagc ccaggccctg gagggctccccaacccagct gcccatcctg 960 gctgacatgc tactctacta ctgccgcttt gccgccagaccggtgctgct gcaagtctat 1020 cagaccgagc tgaccttcat cactggggag aagacgacagagatcttcat ccactccttg 1080 gagctgggtc actccgctgc cacacgtgcc atcaaggcgtcaggtcctgg cagcaagcgg 1140 ctgggcatcg atggcgaccg ggaggctgtt cctctaacactacagattat ttacagccag 1200 ggggccatca gtggacgaag tcgctggagc aacctggagaaggtctgtac ctccgtgaac 1260 ctcaacaagg cctgccggaa gcaggaggag ctggattccagcatggaggc cctgacgcta 1320 aacctgacag aagtggtgaa aaggcagaac tccaaatccaagaagggctt taaccagatt 1380 agcacatcgc agatcaaagt ggacaaggtg cagatcatcggctccaacag ctgccccttt 1440 gctgtgtgcc tggaccagga tgagagaaag atcctgcagagtgtagtcag atgtgaggtc 1500 tcaccgtgct acaagccaga gaagagcgac ctctcctcaccaccccagac gcctcctgac 1560 ctgccggccc aggccgcacc tgatctctgc tccctcctctgcctgcccat catgactttc 1620 agtggagctc tgccctagtg tgggcccagc gccagactggacagaagccc tggggtcatt 1680 tctccagcac taaaatggag tggagagttg gggtggaaataagacatcct taaaaggtta 1740 aattgtctgc aaagcaccta gcccagtgcc gagctcccagtaggtgttca gtaaagctta 1800 gtgcctgact ttctgaacac tgattcctcc tgtttggagtcactgggata ctctcattgc 1860 cgttgggatg ttcctcactc cttcccagtt cgtggctgaggcagaaccca gactgaagag 1920 ggaagagaca ttccagagga ggattgcctt cgtcagggtaaggggtgggc tgctcagggg 1980 ccctaccctt cacccccttc tgtatcagat tggccctcccactcccatct cactctgcgt 2040 gtacaatctt ccatatccgc aagttcactg gcactcttctggcacctggg caagatccca 2100 gaacagagga tggagtgact ggcctcacag agcttagtgcccgacactgg tgcatgggaa 2160 atggtcagcc taggatagga cacgagagtc tgaaattcaaagcaaccagc ttgaagtggt 2220 ttgagaagct ggaagcaaac atgggctaga gagatagggcagaagtcaag acgaggatct 2280 ggactgatgt ggagaaagta gccacggaag catgaactgtatcctgcaca aagtccctct 2340 tccccgcctc ctaattcatt atgcccaaaa ggccttacgtgaaattccag cccagagtac 2400 tcatgacttg agagacgtgg acagagccag cttctaccttgcctggccgt ctctcccctg 2460 tcttaatgtc tgctcttgct ctaagctcca gaagagtggcgggccatgta tcttcaatat 2520 gtttttgctg tatgggcagg ttgtcttatt atgtgatcaacagatgtcca ggaactaatg 2580 agtggaattt aatattattg tcaaataaaa cttgatttgtcctat 2625 2 447 PRT HOMO SAPIENS 2 Met Gln Pro Gly Ala Thr Thr Cys ThrGlu Asp Arg Ile Gln His Ala 1 5 10 15 Leu Glu Arg Cys Leu His Gly LeuSer Leu Ser Arg Arg Ser Thr Ser 20 25 30 Trp Ser Ala Gly Leu Cys Leu AsnCys Trp Ser Leu Gln Glu Leu Val 35 40 45 Ser Arg Asp Pro Gly His Phe LeuIle Leu Leu Glu Gln Ile Leu Gln 50 55 60 Lys Thr Arg Glu Val Gln Glu LysGly Thr Tyr Asp Leu Leu Thr Pro 65 70 75 80 Leu Ala Leu Leu Phe Tyr SerThr Val Leu Cys Thr Pro His Phe Pro 85 90 95 Pro Asp Ser Asp Leu Leu LeuLys Ala Ala Ser Thr Tyr His Arg Phe 100 105 110 Leu Thr Trp Pro Val ProTyr Cys Ser Ile Cys Gln Glu Leu Leu Thr 115 120 125 Phe Ile Asp Ala GluLeu Lys Ala Pro Gly Ile Ser Tyr Gln Arg Leu 130 135 140 Val Arg Ala GluGln Gly Leu Pro Ile Arg Ser His Arg Ser Ser Thr 145 150 155 160 Glu LeuGly Thr Thr Pro Trp Glu Glu Ser Thr Asn Gly Ile Ser His 165 170 175 TyrLeu Gly Met Leu Asp Pro Trp Tyr Glu Arg Asn Val Leu Gly Leu 180 185 190Met His Leu Pro Pro Glu Val Leu Cys Gln Gln Ser Leu Lys Ala Glu 195 200205 Ala Gln Ala Leu Glu Gly Ser Pro Thr Gln Leu Pro Ile Leu Ala Asp 210215 220 Met Leu Leu Tyr Tyr Cys Arg Phe Ala Ala Arg Pro Val Leu Leu Gln225 230 235 240 Val Tyr Gln Thr Glu Leu Thr Phe Ile Thr Gly Glu Lys ThrThr Glu 245 250 255 Ile Phe Ile His Ser Leu Glu Leu Gly His Ser Ala AlaThr Arg Ala 260 265 270 Ile Lys Ala Ser Gly Pro Gly Ser Lys Arg Leu GlyIle Asp Gly Asp 275 280 285 Arg Glu Ala Val Pro Leu Thr Leu Gln Ile IleTyr Ser Gln Gly Ala 290 295 300 Ile Ser Gly Arg Ser Arg Trp Ser Asn LeuGlu Lys Val Cys Thr Ser 305 310 315 320 Val Asn Leu Asn Lys Ala Cys ArgLys Gln Glu Glu Leu Asp Ser Ser 325 330 335 Met Glu Ala Leu Thr Leu AsnLeu Thr Glu Val Val Lys Arg Gln Asn 340 345 350 Ser Lys Ser Lys Lys GlyPhe Asn Gln Ile Ser Thr Ser Gln Ile Lys 355 360 365 Val Asp Lys Val GlnIle Ile Gly Ser Asn Ser Cys Pro Phe Ala Val 370 375 380 Cys Leu Asp GlnAsp Glu Arg Lys Ile Leu Gln Ser Val Val Arg Cys 385 390 395 400 Glu ValSer Pro Cys Tyr Lys Pro Glu Lys Ser Asp Leu Ser Ser Pro 405 410 415 ProGln Thr Pro Pro Asp Leu Pro Ala Gln Ala Ala Pro Asp Leu Cys 420 425 430Ser Leu Leu Cys Leu Pro Ile Met Thr Phe Ser Gly Ala Leu Pro 435 440 4453 3040 DNA HOMO SAPIENS 3 ccctttccac ctctctgctc ccattcctga ccccttacttcccacacctc tgtcccgttc 60 tgctgcaggg gtgctctgtc ctgccactca gatgtggccctccagatgcc attcctaccc 120 tggaggcagc tgtaaggccc ctggtcctgt ttccacagcacctgagctat agctgggctg 180 ggctgatcgc gctgcactgt gagcacctgt tgtctttactggaccaggtg ctctctggga 240 aaggagctcg acaagctgac cggcgtctgt cccccatgcaggcgatgacc caggatgcag 300 ccaggggcca cgacatgcac ggaggaccgc atccagcatgccctggaacg ctgcctgcat 360 ggactcagcc tcagccgccg ctccacctcc tggtcagctgggctgtgtct gaactgctgg 420 agcctgcagg agctggtcag cagggacccg ggccacttccttatcctcct tgagcagatc 480 ctgcagaaga cccgagaggt ccaggagaag ggcacctacgacctgctcac cccgctggcc 540 ctgctcttct attccactgt tctttgtaca ccacacttcccaccagactc ggatctcctt 600 ctgaaggcag ccagcaccta ccaccggttc ctgacctggcctgttcctta ctgcagcatc 660 tgccaggagc tgctcacctt cattgatgct gaactcaaggccccaggtat ctcctaccag 720 agactggtga gggctgagca gggcctgccc atcaggagtcaccgcagctc caccgtcacc 780 gtgctgctgc tgaacccagt ggaagtgcag gccgagttccttgctgtagc caataagctg 840 agtacgcccg gacactcgcc tcacagtgcc tacaccaccctgctcctgca cgccttccag 900 gccacctttg gggcccactg tgacgtcccg ggcctgcactgcaggctaca ggccaagacc 960 ctggcagagc ttgaggacat cttcacggag accgcagaggcacaggagct ggcatctggc 1020 atcggggatg ctgcagaggc ccggcggtgg ctcaggaccaagctgcaggc ggtgggagaa 1080 aaagctggct tccctggggt gttagacact gcaaaaccagggaagcttca taccatcccc 1140 atccctgtcg ccaggtgcta cacctacagc tggagccaggacagctttgg agctgggcac 1200 caccccatgg gaggagagca ccaatggcat ctcccactacctcggcatgc tggacccctg 1260 gtatgagcgc aatgtactgg gcctcatgca cctgccccctgaagtcctgt gccagcagtc 1320 cctgaaggct gaagcccagg ccctggaggg ctccccaacccagctgccca tcctggctga 1380 catgctactc tactactgcc gctttgccgc cagaccggtgctgctgcaag tctatcagac 1440 cgagctgacc ttcatcactg gggagaagac gacagagatcttcatccact ccttggagct 1500 gggtcactcc gctgccacac gtgccatcaa ggcgtcaggtcctggcagca agcggctggg 1560 catcgatggc gaccgggagg ctgttcctct aacactacagattatttaca gccagggggc 1620 catcagtgga cgaagtcgct ggagcaacct ggagaaggtctgtacctccg tgaacctcaa 1680 caaggcctgc cggaagcagg aggagctgga ttccagcatggaggccctga cgctaaacct 1740 gacagaagtg gtgaaaaggc agaactccaa atccaagaagggctttaacc agattagcac 1800 atcgcagatc aaagtggaca aggtgcagat catcggctccaacagctgcc cctttgctgt 1860 gtgcctggac caggatgaga gaaagatcct gcagagtgtagtcagatgtg aggtctcacc 1920 gtgctacaag ccagagaaga gcgacctctc ctcaccaccccagacgcctc ctgacctgcc 1980 ggcccaggcc gcacctgatc tctgctccct cctctgcctgcccatcatga ctttcagtgg 2040 agctctgccc tagtgtgggc ccagcgccag actggacagaagccctgggg tcatttctcc 2100 agcactaaaa tggagtggag agttggggtg gaaataagacatccttaaaa ggttaaattg 2160 tctgcaaagc acctagccca gtgccgagct cccagtaggtgttcagtaaa gcttagtgcc 2220 tgactttctg aacactgatt cctcctgttt ggagtcactgggatactctc attgccgttg 2280 ggatgttcct cactccttcc cagttcgtgg ctgaggcagaacccagactg aagagggaag 2340 agacattcca gaggaggatt gccttcgtca gggtaaggggtgggctgctc aggggcccta 2400 cccttcaccc ccttctgtat cagattggcc ctcccactcccatctcactc tgcgtgtaca 2460 atcttccata tccgcaagtt cactggcact cttctggcacctgggcaaga tcccagaaca 2520 gaggatggag tgactggcct cacagagctt agtgcccgacactggtgcat gggaaatggt 2580 cagcctagga taggacacga gagtctgaaa ttcaaagcaaccagcttgaa gtggtttgag 2640 aagctggaag caaacatggg ctagagagat agggcagaagtcaagacgag gatctggact 2700 gatgtggaga aagtagccac ggaagcatga actgtatcctgcacaaagtc cctcttcccc 2760 gcctcctaat tcattatgcc caaaaggcct tacgtgaaattccagcccag agtactcatg 2820 acttgagaga cgtggacaga gccagcttct accttgcctggccgtctctc ccctgtctta 2880 atgtctgctc ttgctctaag ctccagaaga gtggcgggccatgtatcttc aatatgtttt 2940 tgctgtatgg gcaggttgtc ttattatgtg atcaacagatgtccaggaac taatgagtgg 3000 aatttaatat tattgtcaaa taaaacttga tttgtcctat3040 4 323 PRT HOMO SAPIENS 4 Met Gln Pro Gly Ala Thr Thr Cys Thr GluAsp Arg Ile Gln His Ala 1 5 10 15 Leu Glu Arg Cys Leu His Gly Leu SerLeu Ser Arg Arg Ser Thr Ser 20 25 30 Trp Ser Ala Gly Leu Cys Leu Asn CysTrp Ser Leu Gln Glu Leu Val 35 40 45 Ser Arg Asp Pro Gly His Phe Leu IleLeu Leu Glu Gln Ile Leu Gln 50 55 60 Lys Thr Arg Glu Val Gln Glu Lys GlyThr Tyr Asp Leu Leu Thr Pro 65 70 75 80 Leu Ala Leu Leu Phe Tyr Ser ThrVal Leu Cys Thr Pro His Phe Pro 85 90 95 Pro Asp Ser Asp Leu Leu Leu LysAla Ala Ser Thr Tyr His Arg Phe 100 105 110 Leu Thr Trp Pro Val Pro TyrCys Ser Ile Cys Gln Glu Leu Leu Thr 115 120 125 Phe Ile Asp Ala Glu LeuLys Ala Pro Gly Ile Ser Tyr Gln Arg Leu 130 135 140 Val Arg Ala Glu GlnGly Leu Pro Ile Arg Ser His Arg Ser Ser Thr 145 150 155 160 Val Thr ValLeu Leu Leu Asn Pro Val Glu Val Gln Ala Glu Phe Leu 165 170 175 Ala ValAla Asn Lys Leu Ser Thr Pro Gly His Ser Pro His Ser Ala 180 185 190 TyrThr Thr Leu Leu Leu His Ala Phe Gln Ala Thr Phe Gly Ala His 195 200 205Cys Asp Val Pro Gly Leu His Cys Arg Leu Gln Ala Lys Thr Leu Ala 210 215220 Glu Leu Glu Asp Ile Phe Thr Glu Thr Ala Glu Ala Gln Glu Leu Ala 225230 235 240 Ser Gly Ile Gly Asp Ala Ala Glu Ala Arg Arg Trp Leu Arg ThrLys 245 250 255 Leu Gln Ala Val Gly Glu Lys Ala Gly Phe Pro Gly Val LeuAsp Thr 260 265 270 Ala Lys Pro Gly Lys Leu His Thr Ile Pro Ile Pro ValAla Arg Cys 275 280 285 Tyr Thr Tyr Ser Trp Ser Gln Asp Ser Phe Gly AlaGly His His Pro 290 295 300 Met Gly Gly Glu His Gln Trp His Leu Pro LeuPro Arg His Ala Gly 305 310 315 320 Pro Leu Val 5 3630 DNA HOMO SAPIENS5 atgcagccag gggccacgac atgcacggag gaccgcatcc agcatgccct ggaacgctgc 60ctgcatggac tcagcctcag ccgccgctcc acctcctggt cagctgggct gtgtctgaac 120tgctggagcc tgcaggagct ggtcagcagg gacccgggcc acttccttat cctccttgag 180cagatcctgc agaagacccg agaggtccag gagaagggca cctacgacct gctcaccccg 240ctggccctgc tcttctattc cactgttctt tgtacaccac acttcccacc agactcggat 300ctccttctga aggcagccag cacctaccac cggttcctga cctggcctgt tccttactgc 360agcatctgcc aggagctgct caccttcatt gatgctgaac tcaaggcccc agggatctcc 420taccagagac tggtgagggc tgagcagggc ctgcccatca ggagtcaccg cagctccacc 480gtcaccgtgc tgctgctgaa cccagtggaa gtgcaggccg agttccttgc tgtagccaat 540aagctgagta cgcccggaca ctcgcctcac agtgcctaca ccaccctgct cctgcacgcc 600ttccaggcca cctttggggc ccactgtgac gtcccgggcc tgcactgcag gctacaggcc 660aagaccctgg cagagcttga ggacatcttc acggagaccg cagaggcaca ggagctggca 720tctggcatcg gggatgctgc agaggcccgg cggtggctca ggaccaagct gcaggcggtg 780ggagaaaaag ctggcttccc tggggtgtta gacactgcaa aaccagggaa gcttcatacc 840atccccatcc ctgtcgccag gtgctacacc tacagctgga gccaggacag ctttgacatc 900ctgcaggaaa tcctgctcaa ggaacaggag ctactccagc cagggatcct gggagatgat 960gaagaggagg aagaggagga ggaggaggtg gaggaggact tggaaactga cgggcactgt 1020gccgagagag attccctgct ctccaccagc tctttggcgt cccatgactc caccttgtcc 1080cttgcatcct cccaggcctc ggggccggcc ctctcgcgcc atctgctgac ttcctttgtc 1140tcaggcctct ctgatggcat ggacagcggc tacgtggagg acagcgagga gagctcctcc 1200gagtggcctt ggaggcgtgg cagccaggaa cgccgaggcc accgcaggcc tgggcagaag 1260ttcatcagga tctataaact cttcaagagc accagccagc tggtactgcg gagggactct 1320cggagcctgg agggcagctc ggacacggcc ctgcccctga ggcgggcagg gagcctctgc 1380agccccctgg acgaaccagt atcaccccct tcccgggccc agcgctcccg ctccctgccc 1440cagcccaaac tcggtaccca gctgcccagc tggcttctgg cccctgcttc acgcccccag 1500cgccgccgcc ccttcctgag tggagatgag gatcccaagg cttccacgct acgtgttgtg 1560gtctttggct ccgatcggat ttcagggaag gtggctcggg cgtacagcaa ccttcggcgg 1620ctggagaaca atcgcccact cctcacacgg ttcttcaaac ttcagttctt ctacgtgcct 1680gtgaagcgaa gtcgtgggac cagccctggt gcctgtccac cccctcggag ccagacgccc 1740tcacccccga cagactcccc taggcacgcc agccctggag agctgggcac caccccatgg 1800gaggagagca ccaatggcat ctcccactac ctcggcatgc tggacccctg gtatgagcgc 1860aatgtactgg gcctcatgca cctgccccct gaagtcctgt gccagcagtc cctgaaggct 1920gaagcccagg ccctggaggg ctccccaacc cagctgccca tcctggctga catgctactc 1980tactactgcc gctttgccgc cagaccggtg ctgctgcaag tctatcagac cgagctgacc 2040ttcatcactg gggagaagac gacagagatc ttcatccact ccttggagct gggtcactcc 2100gctgccacac gtgccatcaa ggcgtcaggt cctggcagca agcggctggg catcgatggc 2160gaccgggagg ctgttcctct aacactacag attatttaca gccagggggc catcagtgga 2220cgaagtcgct ggagcaacct ggagaaggtc tgtacctccg tgaacctcaa caaggcctgc 2280cggaagcagg aggagctgga ttccagcatg gaggccctga cgctaaacct gacagaagtg 2340gtgaaaaggc agaactccaa atccaagaag ggctttaacc agattagcac atcgcagatc 2400aaagtggaca aggtgcagat catcggctcc aacagctgcc cctttgctgt gtgcctggac 2460caggatgaga gaaagatcct gcagagtgta gtcagatgtg aggtctcacc gtgctacaag 2520ccagagaaga gcgacctctc ctcaccaccc cagacgcctc ctgacctgcc ggcccaggcc 2580gcacctgatc tctgctccct cctctgcctg cccatcatga ctttcagtgg agctctgccc 2640tagtgtgggc ccagcgccag actggacaga agccctgggg tcatttctcc agcactaaaa 2700tggagtggag agttggggtg gaaataagac atccttaaaa ggttaaattg tctgcaaagc 2760acctagccca gtgccgagct cccagtaggt gttcagtaaa gcttagtgcc tgactttctg 2820aacactgatt cctcctgttt ggagtcactg ggatactctc attgccgttg ggatgttcct 2880cactccttcc cagttcgtgg ctgaggcaga acccagactg aagagggaag agacattcca 2940gaggaggatt gccttcgtca gggtaagggg tgggctgctc aggggcccta cccttcaccc 3000ccttctgtat cagattggcc ctcccactcc catctcactc tgcgtgtaca atcttccata 3060tccgcaagtt cactggcact cttctggcac ctgggcaaga tcccagaaca gaggatggag 3120tgactggcct cacagagctt agtgcccgac actggtgcat gggaaatggt cagcctagga 3180taggacacga gagtctgaaa ttcaaagcaa ccagcttgaa gtggtttgag aagctggaag 3240caaacatggg ctagagagat agggcagaag tcaagacgag gatctggact gatgtggaga 3300aagtagccac ggaagcatga actgtatcct gcacaaagtc cctcttcccc gcctcctaat 3360tcattatgcc caaaaggcct tacgtgaaat tccagcccag agtactcatg acttgagaga 3420cgtggacaga gccagcttct accttgcctg gccgtctctc ccctgtctta atgtctgctc 3480ttgctctaag ctccagaaga gtggcgggcc atgtatcttc aatatgtttt tgctgtatgg 3540gcaggttgtc ttattatgtg atcaacagat gtccaggaac taatgagtgg aatttaatat 3600tattgtcaaa taaaacttga tttgtcctat 3630 6 880 PRT HOMO SAPIENS 6 Met GlnPro Gly Ala Thr Thr Cys Thr Glu Asp Arg Ile Gln His Ala 1 5 10 15 LeuGlu Arg Cys Leu His Gly Leu Ser Leu Ser Arg Arg Ser Thr Ser 20 25 30 TrpSer Ala Gly Leu Cys Leu Asn Cys Trp Ser Leu Gln Glu Leu Val 35 40 45 SerArg Asp Pro Gly His Phe Leu Ile Leu Leu Glu Gln Ile Leu Gln 50 55 60 LysThr Arg Glu Val Gln Glu Lys Gly Thr Tyr Asp Leu Leu Thr Pro 65 70 75 80Leu Ala Leu Leu Phe Tyr Ser Thr Val Leu Cys Thr Pro His Phe Pro 85 90 95Pro Asp Ser Asp Leu Leu Leu Lys Ala Ala Ser Thr Tyr His Arg Phe 100 105110 Leu Thr Trp Pro Val Pro Tyr Cys Ser Ile Cys Gln Glu Leu Leu Thr 115120 125 Phe Ile Asp Ala Glu Leu Lys Ala Pro Gly Ile Ser Tyr Gln Arg Leu130 135 140 Val Arg Ala Glu Gln Gly Leu Pro Ile Arg Ser His Arg Ser SerThr 145 150 155 160 Val Thr Val Leu Leu Leu Asn Pro Val Glu Val Gln AlaGlu Phe Leu 165 170 175 Ala Val Ala Asn Lys Leu Ser Thr Pro Gly His SerPro His Ser Ala 180 185 190 Tyr Thr Thr Leu Leu Leu His Ala Phe Gln AlaThr Phe Gly Ala His 195 200 205 Cys Asp Val Pro Gly Leu His Cys Arg LeuGln Ala Lys Thr Leu Ala 210 215 220 Glu Leu Glu Asp Ile Phe Thr Glu ThrAla Glu Ala Gln Glu Leu Ala 225 230 235 240 Ser Gly Ile Gly Asp Ala AlaGlu Ala Arg Arg Trp Leu Arg Thr Lys 245 250 255 Leu Gln Ala Val Gly GluLys Ala Gly Phe Pro Gly Val Leu Asp Thr 260 265 270 Ala Lys Pro Gly LysLeu His Thr Ile Pro Ile Pro Val Ala Arg Cys 275 280 285 Tyr Thr Tyr SerTrp Ser Gln Asp Ser Phe Asp Ile Leu Gln Glu Ile 290 295 300 Leu Leu LysGlu Gln Glu Leu Leu Gln Pro Gly Ile Leu Gly Asp Asp 305 310 315 320 GluGlu Glu Glu Glu Glu Glu Glu Glu Val Glu Glu Asp Leu Glu Thr 325 330 335Asp Gly His Cys Ala Glu Arg Asp Ser Leu Leu Ser Thr Ser Ser Leu 340 345350 Ala Ser His Asp Ser Thr Leu Ser Leu Ala Ser Ser Gln Ala Ser Gly 355360 365 Pro Ala Leu Ser Arg His Leu Leu Thr Ser Phe Val Ser Gly Leu Ser370 375 380 Asp Gly Met Asp Ser Gly Tyr Val Glu Asp Ser Glu Glu Ser SerSer 385 390 395 400 Glu Trp Pro Trp Arg Arg Gly Ser Gln Glu Arg Arg GlyHis Arg Arg 405 410 415 Pro Gly Gln Lys Phe Ile Arg Ile Tyr Lys Leu PheLys Ser Thr Ser 420 425 430 Gln Leu Val Leu Arg Arg Asp Ser Arg Ser LeuGlu Gly Ser Ser Asp 435 440 445 Thr Ala Leu Pro Leu Arg Arg Ala Gly SerLeu Cys Ser Pro Leu Asp 450 455 460 Glu Pro Val Ser Pro Pro Ser Arg AlaGln Arg Ser Arg Ser Leu Pro 465 470 475 480 Gln Pro Lys Leu Gly Thr GlnLeu Pro Ser Trp Leu Leu Ala Pro Ala 485 490 495 Ser Arg Pro Gln Arg ArgArg Pro Phe Leu Ser Gly Asp Glu Asp Pro 500 505 510 Lys Ala Ser Thr LeuArg Val Val Val Phe Gly Ser Asp Arg Ile Ser 515 520 525 Gly Lys Val AlaArg Ala Tyr Ser Asn Leu Arg Arg Leu Glu Asn Asn 530 535 540 Arg Pro LeuLeu Thr Arg Phe Phe Lys Leu Gln Phe Phe Tyr Val Pro 545 550 555 560 ValLys Arg Ser Arg Gly Thr Ser Pro Gly Ala Cys Pro Pro Pro Arg 565 570 575Ser Gln Thr Pro Ser Pro Pro Thr Asp Ser Pro Arg His Ala Ser Pro 580 585590 Gly Glu Leu Gly Thr Thr Pro Trp Glu Glu Ser Thr Asn Gly Ile Ser 595600 605 His Tyr Leu Gly Met Leu Asp Pro Trp Tyr Glu Arg Asn Val Leu Gly610 615 620 Leu Met His Leu Pro Pro Glu Val Leu Cys Gln Gln Ser Leu LysAla 625 630 635 640 Glu Ala Gln Ala Leu Glu Gly Ser Pro Thr Gln Leu ProIle Leu Ala 645 650 655 Asp Met Leu Leu Tyr Tyr Cys Arg Phe Ala Ala ArgPro Val Leu Leu 660 665 670 Gln Val Tyr Gln Thr Glu Leu Thr Phe Ile ThrGly Glu Lys Thr Thr 675 680 685 Glu Ile Phe Ile His Ser Leu Glu Leu GlyHis Ser Ala Ala Thr Arg 690 695 700 Ala Ile Lys Ala Ser Gly Pro Gly SerLys Arg Leu Gly Ile Asp Gly 705 710 715 720 Asp Arg Glu Ala Val Pro LeuThr Leu Gln Ile Ile Tyr Ser Gln Gly 725 730 735 Ala Ile Ser Gly Arg SerArg Trp Ser Asn Leu Glu Lys Val Cys Thr 740 745 750 Ser Val Asn Leu AsnLys Ala Cys Arg Lys Gln Glu Glu Leu Asp Ser 755 760 765 Ser Met Glu AlaLeu Thr Leu Asn Leu Thr Glu Val Val Lys Arg Gln 770 775 780 Asn Ser LysSer Lys Lys Gly Phe Asn Gln Ile Ser Thr Ser Gln Ile 785 790 795 800 LysVal Asp Lys Val Gln Ile Ile Gly Ser Asn Ser Cys Pro Phe Ala 805 810 815Val Cys Leu Asp Gln Asp Glu Arg Lys Ile Leu Gln Ser Val Val Arg 820 825830 Cys Glu Val Ser Pro Cys Tyr Lys Pro Glu Lys Ser Asp Leu Ser Ser 835840 845 Pro Pro Gln Thr Pro Pro Asp Leu Pro Ala Gln Ala Ala Pro Asp Leu850 855 860 Cys Ser Leu Leu Cys Leu Pro Ile Met Thr Phe Ser Gly Ala LeuPro 865 870 875 880 7 696 DNA HOMO SAPIENS 7 caggcgatga cccaggatgcagccaggggc cacgacatgc acggaggacc gcatccagca 60 tgccctggaa cgctgcctgcatggactcag cctcagccgc cgctccacct cctggtcagc 120 tgggctgtgt ctgaactgctggagcctgca ggagctggtc agcagggacc cgggccactt 180 ccttatcctc cttgagcagatcctgcagaa gacccgagag gtccaggaga agggcaccta 240 cgacctgctc accccgctggccctgctctt ctattccact gttctttgta caccacactt 300 cccaccagac tcggatctccttctgaaggc agccagcacc taccaccggt tcctgacctg 360 gcctgttcct tactgcagcatctgccagga gctgctcacc ttcattgatg ctgaactcaa 420 ggccccaggt atctcctaccagagactggt gagggctgag cagggcctgc ccatcaggag 480 tcaccgcagc tccaccaggcctgtccaccc cctcggagcc agacgccctc acccccgaca 540 gactccccta ggcacgccagccctggagag ctgggcacca ccccatggga ggagagcacc 600 aatggcatct cccactacctcggcatgctg gacccctggt atgagcgcaa tgtactgggc 660 ctcatgcacc tgccccctgaagtcctgtgc cagcag 696 8 226 PRT HOMO SAPIENS 8 Met Gln Pro Gly Ala ThrThr Cys Thr Glu Asp Arg Ile Gln His Ala 1 5 10 15 Leu Glu Arg Cys LeuHis Gly Leu Ser Leu Ser Arg Arg Ser Thr Ser 20 25 30 Trp Ser Ala Gly LeuCys Leu Asn Cys Trp Ser Leu Gln Glu Leu Val 35 40 45 Ser Arg Asp Pro GlyHis Phe Leu Ile Leu Leu Glu Gln Ile Leu Gln 50 55 60 Lys Thr Arg Glu ValGln Glu Lys Gly Thr Tyr Asp Leu Leu Thr Pro 65 70 75 80 Leu Ala Leu LeuPhe Tyr Ser Thr Val Leu Cys Thr Pro His Phe Pro 85 90 95 Pro Asp Ser AspLeu Leu Leu Lys Ala Ala Ser Thr Tyr His Arg Phe 100 105 110 Leu Thr TrpPro Val Pro Tyr Cys Ser Ile Cys Gln Glu Leu Leu Thr 115 120 125 Phe IleAsp Ala Glu Leu Lys Ala Pro Gly Ile Ser Tyr Gln Arg Leu 130 135 140 ValArg Ala Glu Gln Gly Leu Pro Ile Arg Ser His Arg Ser Ser Thr 145 150 155160 Arg Pro Val His Pro Leu Gly Ala Arg Arg Pro His Pro Arg Gln Thr 165170 175 Pro Leu Gly Thr Pro Ala Leu Glu Ser Trp Ala Pro Pro His Gly Arg180 185 190 Arg Ala Pro Met Ala Ser Pro Thr Thr Ser Ala Cys Trp Thr ProGly 195 200 205 Met Ser Ala Met Tyr Trp Ala Ser Cys Thr Cys Pro Leu LysSer Cys 210 215 220 Ala Ser 225 9 752 DNA HOMO SAPIENS 9 acaccacacttcccaccaga ctcggatctc cttctgaagg cagccagcac ctaccaccgg 60 ttcctgacctggcctgttcc ttactgcagc atctgccagg agctgctcac cttcattgat 120 gctgaactcaaggccccagg gatctcctac cagagactgg tgagggctga gcagggcctg 180 cccatcaggagtcaccgcag ctccaccgtc accgtgctgc tgctgaaccc agtggaagtg 240 caggccgagttccttgctgt agccaataag ctgagtacgc ccggacactc gcctcacagt 300 gcctacaccaccctgctcct gcacgccttc caggccacct ttggggccca ctgtgacgtc 360 ccgggcctgcactgcaggct acaggccaag accctggcag agcttgagga catcttcacg 420 gagaccgcagaggcacagga gctggcatct ggcatcgggg atgctgcaga ggcccggcgg 480 tggctcaggaccaagctgca ggcggtggga gaaaaagctg gcttccctgg ggtgttagac 540 actgcaaaaccagggaagct tcataccatc cccatccctg tcgccaggtg ctacacctac 600 agctggagccaggacagctt tgggagctgg gcaccacccc atgggaggag agcaccaatg 660 gcatctcccactacctcggc atgctggacc cctggtatga gcgcaatgta ctgggcctca 720 tgcacctgccccctgaagtc ctgtgccagc ag 752 10 250 PRT HOMO SAPIENS 10 Thr Pro His PhePro Pro Asp Ser Asp Leu Leu Leu Lys Ala Ala Ser 1 5 10 15 Thr Tyr HisArg Phe Leu Thr Trp Pro Val Pro Tyr Cys Ser Ile Cys 20 25 30 Gln Glu LeuLeu Thr Phe Ile Asp Ala Glu Leu Lys Ala Pro Gly Ile 35 40 45 Ser Tyr GlnArg Leu Val Arg Ala Glu Gln Gly Leu Pro Ile Arg Ser 50 55 60 His Arg SerSer Thr Val Thr Val Leu Leu Leu Asn Pro Val Glu Val 65 70 75 80 Gln AlaGlu Phe Leu Ala Val Ala Asn Lys Leu Ser Thr Pro Gly His 85 90 95 Ser ProHis Ser Ala Tyr Thr Thr Leu Leu Leu His Ala Phe Gln Ala 100 105 110 ThrPhe Gly Ala His Cys Asp Val Pro Gly Leu His Cys Arg Leu Gln 115 120 125Ala Lys Thr Leu Ala Glu Leu Glu Asp Ile Phe Thr Glu Thr Ala Glu 130 135140 Ala Gln Glu Leu Ala Ser Gly Ile Gly Asp Ala Ala Glu Ala Arg Arg 145150 155 160 Trp Leu Arg Thr Lys Leu Gln Ala Val Gly Glu Lys Ala Gly PhePro 165 170 175 Gly Val Leu Asp Thr Ala Lys Pro Gly Lys Leu His Thr IlePro Ile 180 185 190 Pro Val Ala Arg Cys Tyr Thr Tyr Ser Trp Ser Gln AspSer Phe Gly 195 200 205 Ser Trp Ala Pro Pro His Gly Arg Arg Ala Pro MetAla Ser Pro Thr 210 215 220 Thr Ser Ala Cys Trp Thr Pro Gly Met Ser AlaMet Tyr Trp Ala Ser 225 230 235 240 Cys Thr Cys Pro Leu Lys Ser Cys AlaSer 245 250

What is claimed is:
 1. An expression system comprising a polynucleotideencoding a polypeptide comprising the amino acid sequence set forth inSEQ ID NO:2, wherein said expression system is capable of producing saidpolypeptide when said expression system is present in a compatible hostcell.
 2. A process for producing a recombinant host cell which comprisesthe step of introducing an expression vector comprising a polynucleotideencoding SEQ ID NO:2.
 3. A recombinant host cell produced by the processof claim
 2. 4. A membrane of a recombinant host cell of claim 3expressing said polypeptide.
 5. A process for producing a polypeptidewhich comprises culturing a host cell of claim 3 under conditionssufficient for the production of said polypeptide and recovering saidpolypeptide from the culture.
 6. An expression system comprising apolynucleotide encoding a polypeptide comprising the amino acid sequenceset forth in SEQ ID NO:4, wherein said expression system is capable ofproducing said polypeptide when said expression system is present in acompatible host cell.
 7. A process for producing a recombinant host cellwhich comprises the step of introducing into a host cell an expressionvector comprising a polynucleotide encoding SEQ ID NO:4.
 8. Arecombinant host cell produced by the process of claim
 7. 9. A membraneof a recombinant host cell of claim 8 expressing said polypeptide.
 10. Aprocess of producing a polypeptide which comprises culturing a host cellof claim 8 under conditions sufficient for the production of saidpolypeptide and recovering said polypeptide from culture.
 11. Anisolated polynucleotide comprising a polynucleotide sequence that has atleast 95% identity to the polynucleotide sequence set forth in SEQ IDNO:1.
 12. The isolated polynucleotide of claim 11 comprising thenucleotide sequence set forth in SEQ ID NO:1.
 13. The isolatedpolynucleotide of claim 11 consisting of the polynucleotide set forth inSEQ ID NO:
 1. 14. An isolated polynucleotide comprising a polynucleotidesequence encoding a polypeptide that has at least 95% identity to theamino acid sequence set forth in SEQ ID NO:2, wherein said polypeptidehas p101 regulatory activity towards phosphatidylinositol 3-kinase. 15.The isolated polynucleotide of claim 14 comprising a polynucleotideencoding the polypeptide set forth in SEQ ID NO:2.
 16. The isolatedpolynucleotide of claim 14 consisting of an isolated polynucleotideencoding the polypeptide set forth in SEQ ID NO:2.
 17. The isolatedpolynucleotide which is fully complementary to the nucleotide sequencein any one of claims 11-16 over the entire length of said nucleotidesequence set forth in such claim.
 18. An isolated polynucleotidecomprising a polynucleotide sequence that has at least 95% identity tothe polynucleotide sequence set forth in SEQ ID NO:3, wherein saidpolynucleotide encodes a polypeptide having p101 regulatory activitytowards phosphatidylinositol 3-kinase.
 19. The isolated polynucleotideof claim 18 comprising the nucleotide sequence set forth in SEQ ID NO:3.20. The isolated polynucleotide of claim 18 consisting of thepolynucleotide set forth in SEQ ID NO:3.
 21. An isolated polynucleotidecomprising a polynucleotide sequence encoding a polypeptide that has atleast 95% identity to the amino acid sequence set forth in SEQ ID NO:4,wherein said polypeptide has p101 regulatory activity towardsphosphatidylinositol 3-kinase.
 22. The isolated polynucleotide of claim21 comprising a polynucleotide encoding the polypeptide set forth in SEQID NO:4.
 23. The isolated polynucleotide of claim 21 consisting of anisolated polynucleotide encoding the polypeptide set forth in SEQ IDNO:4.
 24. An isolated polynucleotide which is fully complementary to thenucleotide sequence in any one of claims 18-23 over the entire length ofsaid nucleotide sequence set forth in such claim.